Nucleic acid polymerase variants, kits and methods for template-independent rna synthesis

ABSTRACT

Provided herein relates to nucleic acid polymerase variants and kits including the same, where the nucleic acid polymerase variant has an improved function and activity of performing template-independent nucleic acids synthesis using ribonucleotides (rNTPs) in a thermotolerant manner.

CROSS REFERENCE

This application claims priority to, and the benefit of, U.S.Provisional Application No. US63/249,819, filed on Sep. 29, 2021, thecontent thereof is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to nucleic acid polymerase variants andkits comprising the same for use particularly in the context of de novoenzymatic nucleic acid synthesis.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledP222338US-sequence_listing, created on Sep. 28, 2022, which is 96 kb insize. The information in the electronic format of Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND

Synthetic oligonucleotides are crucial to many aspects ofbiotechnological research in both the academic and industrial settings.However, there are many limitations of traditional chemical synthesismethods developed decades ago. This is especially true for de novo RNAsynthesis, which remains largely inaccessible to those heavily investedin advancing genome engineering technologies, RNA-based diagnostics,RNA-based therapeutics, RNA-based sequencing technologies, nucleicacid-based information storage, and even biological computing. Chemicalsynthesis of RNA is troubled by lengthy reaction steps that require bothharsh chemical reagents and biologically incompatible organic solvents.These reaction conditions often lead to the depurination of thenucleotide bases, unexpected insertions or deletions from the overallsequence, and the premature irreversible capping of the oligonucleotideresulting in unwanted truncated products. This substantially increasesthe overall error-rate of RNA synthesis, limits the synthesis length ofRNA oligonucleotides (less than 120 nucleotides), and requires longerlead-times to obtain acceptable yields of a desired product. Moreover,the chemical synthesis of RNA oligonucleotide is toxic andlabor-intensive. Overcome the current limitations of RNA oligonucleotidesynthesis is therefore important.

Enzyme-based de novo nucleic acid synthesis is an emerging, non-toxicmethod to substitute for the decades-old, toxic, chemicalphosphoramidite-based nucleic acid synthesis. All living organisms relyon nucleic acid polymerases to efficiently duplicate their nucleic acid.Owing to their nucleic acid duplication mechanism, most nucleic acidpolymerases require a template to direct synthesis and incorporation ofnucleotides into a growing nucleic acid strand. The template-dependentmanner of nucleic acid synthesis requires the polymerase to associatewith a primer-template nucleic acid before the nucleotide can be addedto the 3′-terminus of primer by the polymerase.

Unlike most replicative nucleic acid polymerases, the X-family terminaldeoxynucleotidyl transferase (Tdt) is a unique class of mesophilicenzyme, which doesn’t rely on a template for adding nucleotides duringnucleic acid synthesis. Tdt only requires a short initiator DNA orprimer to direct synthesis and incorporation of nucleotides into agrowing initiator DNA or primer. Previous studies revealed that Tdt canincorporate both deoxyribonucleotides (dNTPs) and ribonucleotides(rNTPs) with a minor discrimination (Boule, J B et al. The Journal ofbiological chemistry (2001)). However, Tdt fails to extend the newlysynthesized RNA strand beyond around 4-5 ribonucleotides, suggestingthat Tdt has impeded accommodation of ribo- or mixedribo/deoxyribonucleic acid substrates for further synthesis. Therefore,Tdt enzyme is not suitable for de novo RNA synthesis.

SUMMARY OF THE INVENTION

Owing to the diverse structure-function relationships mentioned above,the naturally occurring, replicative nucleic acid polymerases cannotreadily utilize ribonucleotides (rNTPs) as a substrate for de novoribonucleic acid synthesis. Thus, the tailor-made, modified nucleic acidpolymerase is a prerequisite for exerting the utilities of a variety ofnucleic acid synthesis applications.

The inventor has discovered the novel positions/regions in the aminoacid sequences of B-family DNA polymerase variants that play crucialparts in endowing the said polymerases with a template-independence andan enhancing nucleotide substrate binding affinity of said polymerasesfor ribonucleotides, thereby providing a new option for thetemplate-independent RNA synthesis method.

Accordingly, in one aspect, the present disclosure provides a,so-called, RNA polymerase variant comprising: a motif A, and a motif Bcorresponding to positions 706 to 730, and 843 to 855, respectively, ofa consensus sequence (SEQ ID NO:1); and at least one amino acidsubstitution at a position in the motif A, the motif B, or thecombination thereof; wherein the RNA polymerase variant has a reduced oreliminated the intrinsic 3′ to 5′ exonuclease activity.

In one embodiment, the representative RNA polymerase variant is modifiedfrom a wild-type B-family DNA polymerase having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 and 17.

In one embodiment, the wild-type B-family DNA polymerase is Thermococcusgorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNApolymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N),Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNApolymerase (Vent), Methanosarcina acetivorans DNA polymerase (Mac),Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNApolymerase (Sso), Methanococcus maripaludis DNA polymerase (Mma), humanDNA polymerase, delta catalytic p125 subunit (hPOLD), Saccharomycescerevisiae DNA polymerase delta catalytic subunit (ScePOLD), Pseudomonasaeruginosa DNA polymerase II (Pae), Escherichia. coli DNA polymerase II(Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phageT4 DNA polymerase (T4), or Bacillus phage Phi29 DNA polymerase (Phi29).

In one embodiment, the representative RNA polymerase variant comprises amotif Exo I corresponding to positions 349 to 364 of the consensussequence (SEQ ID NO:1), and the RNA polymerase variant has at least oneamino acid substitution at a position in the motif Exo I. Preferably, anamino acid L or M corresponding to position 715 of SEQ ID NO: 1 issubstituted with A, C, D, F, G, H, K, N, Q, S, W, or Y; an amino acid Ycorresponding to position 716 of SEQ ID NO: 1 remains unchanged or issubstituted with A, C, D, G, N, S, T or V; and an amino acid Pcorresponding to position 717 of SEQ ID NO: 1 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the selected RNA polymerase variant is derived fromThermococcus gorgonarius DNA polymerase (Tgo) having a wild-type aminoacid sequence of SEQ ID NO: 2; and wherein: an amino acid L at position408 of SEQ ID NO: 2 is substituted with A, C, D, F, G, H, K, M, N, Q, S,W, or Y; an amino acid Y at position 409 of SEQ ID NO: 2 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; and an aminoacid P at position 410 of SEQ ID NO: 2 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-typeamino acid sequence of SEQ ID NO: 2; and wherein: an amino acid L atposition 408 of SEQ ID NO: 2 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 409 of SEQ ID NO: 2remains unchanged or is substituted with A, C, D, G, N, S, T or V; anamino acid P at position 410 of SEQ ID NO: 2 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and an amino acid A atposition 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-typeamino acid sequence of SEQ ID NO: 3; and wherein: an amino acid L atposition 408 of SEQ ID NO: 3 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 409 of SEQ ID NO: 3remains unchanged or is substituted with A, C, D, G, N, S, T or V; andan amino acid P at position 410 of SEQ ID NO: 3 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-typeamino acid sequence of SEQ ID NO: 3; and wherein: an amino acid L atposition 408 of SEQ ID NO: 3 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 409 of SEQ ID NO: 3remains unchanged or is substituted with A, C, D, G, N, S, T or V; anamino acid P at position 410 of SEQ ID NO: 3 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and an amino acid A atposition 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having awild-type amino acid sequence of SEQ ID NO: 4; and wherein: an aminoacid L at position 408 of SEQ ID NO: 4 is substituted with A, C, D, F,G, H, K, M, N, Q, S, W, or Y; an amino acid Y at position 409 of SEQ IDNO: 4 is remains unchanged or is substituted with A, C, D, G, N, S, T orV; and an amino acid P at position 410 of SEQ ID NO: 4 remains unchangedor is substituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having awild-type amino acid sequence of SEQ ID NO: 4; and wherein: an aminoacid L at position 408 of SEQ ID NO: 4 is substituted with A, C, D, F,G, H, K, M, N, Q, S, W, or Y; an amino acid Y at position 409 of SEQ IDNO: 4 remains unchanged or is substituted with A, C, D, G, N, S, T or V;an amino acid P at position 410 of SEQ ID NO: 4 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and an amino acid A atposition 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant is derivedfrom Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type aminoacid sequence of SEQ ID NO: 5; and wherein: an amino acid L at position409 of SEQ ID NO: 5 is substituted with A, C, D, F, G, H, K, M, N, Q, S,W, or Y; an amino acid Y at position 410 of SEQ ID NO: 5 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; and an aminoacid P at position 411 of SEQ ID NO: 5 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type aminoacid sequence of SEQ ID NO: 5; and wherein: an amino acid L at position409 of SEQ ID NO: 5 is substituted with A, C, D, F, G, H, K, M, N, Q, S,W, or Y; an amino acid Y at position 410 of SEQ ID NO: 5 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; an amino acidP at position 411 of SEQ ID NO: 5 remains unchanged or is substitutedwith A, C, G, I, L, M, N, S, T or V; and an amino acid A at position 486of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, I, K, L, M, N, P,Q, R, T, V, W or Y

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus litoralis DNA polymerase (Vent) having a wild-typeamino acid sequence of SEQ ID NO: 6; and wherein: an amino acid L atposition 411 of SEQ ID NO: 6 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 412 of SEQ ID NO: 6remains unchanged or is substituted with A, C, D, G, N, S, T or V; andan amino acid P at position 413 of SEQ ID NO: 6 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Thermococcus litoralis DNA polymerase (Vent) having a wild-typeamino acid sequence of SEQ ID NO: 6; and wherein: an amino acid L atposition 411 of SEQ ID NO: 6 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 412 of SEQ ID NO: 6remains unchanged or is substituted with A, C, D, G, N, S, T or V; anamino acid P at position 413 of SEQ ID NO: 6 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and an amino acid A atposition 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant is derivedfrom Methanosarcina acetivorans DNA polymerase (Mac) having a wild-typeamino acid sequence of SEQ ID NO: 7; and wherein: an amino acid L atposition 485 of SEQ ID NO: 7 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 486 of SEQ ID NO: 7remains unchanged or is substituted with A, C, D, G, N, S, T or V; andan amino acid P at position 487 of SEQ ID NO: 7 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Methanosarcina acetivorans DNA polymerase (Mac) having a wild-typeamino acid sequence of SEQ ID NO: 7; and wherein: an amino acid L atposition 485 of SEQ ID NO: 7 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 486 of SEQ ID NO: 7remains unchanged or is substituted with A, C, D, G, N, S, T or V; anamino acid P at position 487 of SEQ ID NO: 7 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and an amino acid A atposition 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant is derivedfrom Pyrobaculum islandicum DNA polymerase (Pis) having a wild-typeamino acid sequence of SEQ ID NO: 8; and wherein: an amino acid M atposition 426 of SEQ ID NO: 8 is substituted with A, C, D, F, G, H, K, N,Q, S, W, or Y; an amino acid Y at position 427 of SEQ ID NO: 8 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; and an aminoacid P at position 428 of SEQ ID NO: 8 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Pyrobaculum islandicum DNA polymerase (Pis) having a wild-typeamino acid sequence of SEQ ID NO: 8; and wherein: an amino acid M atposition 426 of SEQ ID NO: 8 is substituted with A, C, D, F, G, H, K, N,Q, S, W, or Y; an amino acid Y at position 427 of SEQ ID NO: 8 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; an amino acidP at position 428 of SEQ ID NO: 8 remains unchanged or is substitutedwith A, C, G, I, L, M, N, S, T or V; and an amino acid A at position 508of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, I, K, L, M, N, P,Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant is derivedfrom Sulfolobus solfataricus DNA polymerase (Sso) having a wild-typeamino acid sequence of SEQ ID NO: 9; and wherein: an amino acid L atposition 518 of SEQ ID NO: 9 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 519 of SEQ ID NO: 9remains unchanged or is substituted with A, C, D, G, N, S, T or V; andan amino acid P at position 520 of SEQ ID NO: 9 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.

In one embodiment, the representative RNA polymerase variant is derivedfrom Sulfolobus solfataricus DNA polymerase (Sso) having a wild-typeamino acid sequence of SEQ ID NO: 9; and wherein: an amino acid L atposition 518 of SEQ ID NO: 9 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; an amino acid Y at position 519 of SEQ ID NO: 9remains unchanged or is substituted with A, C, D, G, N, S, T or V; anamino acid P at position 520 of SEQ ID NO: 9 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and an amino acid A atposition 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y.

In one embodiment, the representative RNA polymerase variant exhibits anactivity of synthesizing nucleic acids in a template-independent mannerby adding at least one nucleotide selected from the group of naturallyoccurring nucleotide, nucleotide analogue, or a mixture thereof, to anextendible initiator.

In one embodiment, the extendible initiator comprises a single-strandedoligonucleotide initiator, a blunt-ended double-stranded oligonucleotideinitiator, or a mixture thereof.

In one embodiment, the extendible initiator is a free form nucleic acidto be reacted in a liquid phase.

In one embodiment, the extendible initiator is immobilized on a solidsupport, wherein the solid support comprises a particle, bead, slide,array surface, membrane, flow cell, well, microwell, nano-well, chamber,microfluidic chamber, channel, or microfluidic channel.

In one embodiment, the at least one nucleotide is linked with adetectable label.

In one embodiment, the at least one nucleotide comprises a ribose.

In one embodiment, the representative RNA polymerase variant exhibitsthe activity at reaction temperatures ranging from 10° C. to 100° C.

In one aspect, the present disclosure further provides a kit forperforming de novo enzymatic nucleic acid synthesis, comprising an RNApolymerase variant derived from a wild-type B-family DNA polymerasehaving an amino acid sequence selected from the group consisting of SEQID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17,wherein the RNA polymerase variant exhibits activity of synthesizingnucleic acids in a template-independent manner by adding at least onenucleotide selected from the group of naturally occurring nucleotide,nucleotide analogue, or a mixture thereof, to an extendible initiator,thereby synthesizing a desired nucleic acid sequence.

In another aspect, the present disclosure further provides method fortemplate-independent synthesis of an RNA oligonucleotide, comprising:

-   (a) providing an initiator oligonucleotide,-   (b) providing an RNA polymerase variant;-   (c) combining the initiator oligonucleotide, the RNA polymerase    variant and one or more nucleotides under conditions sufficient for    the addition of at least one nucleotide to the 3′ end of the    initiator oligonucleotide;

wherein the selected RNA polymerase variant comprising:

a motif A, and a motif B corresponding to positions 706 to 730, and 843to 855, respectively, of a consensus sequence (SEQ ID NO:1); and atleast one amino acid substitution at a position in the motif A, themotif B, or the combination thereof; wherein the RNA polymerase varianthas a reduced or eliminated the intrinsic 3′ to 5′ exonuclease activity.

Accordingly, the present invention relates to the specific RNApolymerase variants that exhibit an improved performance onincorporating a variety of nucleotides for nucleic acid synthesis atvarious reaction temperatures in the absence of nucleic acid template.More particularly, the de novo RNA synthesis method can be efficientlyperformed by said RNA polymerase variants with a broad-spectrum ofnucleotides and nucleotide analogues.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will become more readily appreciated withreference to the following description in conjunction with theaccompanying drawings.

FIG. 1 shows an amino acid sequence alignment of the wild-type B-familyDNA polymerases (PolB) related to the present invention and theirconsensus sequence.

FIGS. 2A and 2B show the results of the RNA synthesis reactionsdescribed in Example 3.1.

FIGS. 3A and 3B show the results of the RNA synthesis reactionsdescribed in Example 3.2.

FIGS. 4A and 4B show the results of the RNA synthesis reactionsdescribed in Example 3.3.

FIGS. 5A and 5B show the results of the RNA synthesis reactionsdescribed in Example 3.4.

FIGS. 6A and 6B show the results of the RNA synthesis reactionsdescribed in Example 3.5.

DETAILED DESCRIPTION Definition

All terms including descriptive or technical terms which are used hereinshould be construed as having meanings that are understandable to one ofordinary skill in the art. However, the terms may have differentmeanings according to an intention of the user, case precedents, or theappearance of new technologies. Also, some terms may be arbitrarilyselected by the applicant, and in this case, the meaning of the selectedterms will be described in detail in the descriptions of the presentdisclosure. Thus, the terms used herein are defined based on the meaningof the terms together with the descriptions throughout thespecification. In addition, the titles and subtitles may be attached tothe description for readability, but these titles do not affect thescope of the present invention.

As used herein, the term “a,” “an,” or “the” includes plural referentsunless expressly and unequivocally limited to one referent. The term“or” is used interchangeably with the term “and/or” unless the contextclearly indicates otherwise.

Also, when a part or a method “includes” or “comprises” a component or astep, respectively, unless there is a particular description contrarythereto, the part or the method can further include other components orother steps, not excluding the others.

As used herein, an “amino acid” refers to any monomer unit that can beincorporated into a peptide, polypeptide, or protein. As used herein,the term “amino acid” includes the following twenty natural orgenetically encoded alpha-amino acids: alanine (Ala or A), arginine (Argor R), asparagine (Asn or N), aspartic acid or aspartate (Asp or D),cysteine (CyS or C), glutamine (Gln or Q), glutamic acid or glutamate(Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile orI), leucine (Leu or L), lysine (Lys or K), methionine (Met or M),phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S),threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), andvaline (Val or V). In cases where “X” residues are undefined, theseshould be defined as “any amino acid”.

The term “functionally equivalent” or “equivalent” is used to describe aspecific B-family DNA polymerase (PolB) variant and any of thederivative RNA polymerase variant provided herein having thesubstitution or mutation that is considered to occur at the amino acidposition in the other PolB, PolB variant according to the sequencealignment, or a reference sequence, which has the same functional orstructural role in the enzyme. The equivalent positions may be definedaccording to homologues, conserved motifs, user-defined, or derived,consensus sequence.

Generally, the homologous PolBs have similar or identical amino acidsequences and functional structure, and thereby the equivalent aminoacid substitution mutations among different PolBs generally occur athomologous amino acid positions. The term “functionally equivalent” or“equivalent” used herein also encompass mutations that are “homologous”or “positionally equivalent” to a given mutation in view of proteinsequence or structural alignment, regardless of the actual function ofthe mutated amino acid. Practically, the “functionally equivalent”,“homologous” and/or “positionally equivalent” amino acid residues ofdifferent polymerases can be identified according to the proteinsequence or structural alignment. Accordingly, a cross-species alignmentwas made on multiple wild-type PolBs, as illustrated in FIG. 1 , and theconsensus sequence (SEQ ID NO: 1) is used as a positional referencesequence.

For example, the substitution of amino acid aspartic acid (D) withalanine (A) at position 141 of the wild-type Thermococcus kodakarensis(Kod1) (D141A) amino acid sequence would be functionally equivalent tothe amino acid substitution mutation D114A at the conserved residue ofwild-type Escherichia coli phage RB69 DNA polymerase (RB69) amino acidsequence. When the positional reference sequence is used to describethese equivalent amino acid substitutions, the functionally equivalentpositions of both amino acid residue 141 of Kod1 and amino acid residue114 of RB69 corresponds to position 354 of the consensus sequence (SEQID NO: 1).

The term “conserved” means the segment of polymerase having the sameamino acid residue in the homologous or equivalent position of differentPolBs from various sources. The term “semi-conserved” used herein refersto the segment of polymerase that has a similar property of amino acidresidue or an identical amino acid residue in the homologous position ofdifferent PolBs from various sources.

The terms “nucleic acid”, “nucleic acid sequence”, “oligonucleotide”,“polynucleotide”, and “nucleic acid fragment” as used herein refer to adeoxyribonucleotide or ribonucleotide sequence in a single-stranded or adouble-stranded form of which the sources and length are not limitedherein; and generally, includes naturally occurring nucleotides orartificial chemical mimics. The term “nucleic acid” as used herein isinterchangeable with the terms including natural or unnatural“oligonucleotide”, “polynucleotide”, “DNA”, “RNA”, “gene”,“complementary DNA” (cDNA), and “messenger RNA” (mRNA) in use.

The “nucleic acid”, “oligonucleotide”, or “polynucleotide” used hereinrefers to a polymer that can be corresponded to a ribose nucleic acid(RNA) or deoxyribose nucleic acid (DNA) polymer, or an analogue thereof.This includes polymers of nucleotides such as RNA and DNA, as well assynthetic forms, modified (e.g., chemically or biochemically modified)forms thereof, and mixed polymers (e.g., including both RNA and DNAsubunits). Exemplary modifications include methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, and thelike), pendentmoieties (e.g., polypeptides), intercalators (e.g.,acridine, psoralen, and the like), chelators, alkylators, and modifiedlinkages (e.g., alpha anomeric nucleic acids and the like). Alsoincluded are synthetic molecules that mimic polynucleotides in theirability to bind to a designated sequence via hydrogen bonding and otherchemical interactions. Typically, the nucleotide monomers are linked viaphosphodiester bonds, although synthetic forms of nucleic acids cancomprise other linkages (e.g., peptide nucleic acids as described inNielsen et al. Science 254:1497-1500, 1991). A nucleic acid can be orcan include, e.g., a chromosome or chromosomal segment, a vector (e.g.,an expression vector), an expression cassette, a naked DNA or RNApolymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, and a primer. A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence optionally comprises or encodes complementarysequences, in addition to any sequence explicitly indicated.

The nucleic acid as used herein also includes nucleic acid analogue. Theterm nucleic acid analogue is known to describe compounds or artificialnucleic acids which are functionally or structurally equivalent tonaturally existing RNA and DNA. A nucleic acid analogue may have one ormore parts of a nucleotide (the phosphate backbone, pentose sugar, andnucleobase) being modified. These modifications on the nucleotide changethe structure and geometry of the nucleic acid and its interactions withnucleic acid polymerases. The nucleic acid analogue also encompasses theemerging category of artificial nucleic acids, such as XNA, which isdesigned to have new-to-nature forms of sugar backbone.

Examples of nucleic acid analogues include but are not limited to: theuniversal bases, such as inosine, 3-nitropyrrole, and 5-nitroindole,which can form a base-pair with all four canonical bases; thephosphate-sugar backbone analogues, such as peptide-nucleic acids (PNA),which affect the backbone properties of the nucleic acid; chemicallinker or fluorophore-attached analogues, such as amine-reactiveaminoallyl nucleotide, thiol-containing nucleotides, biotin-linkednucleotides, rhodamine-linked nucleotides, and cyanine-linkednucleotides; the fluorescent base analogues, such as 2-aminopurine(2-AP), 3-methylisoxanthopterin (3-MI), 3-methylisoxanthopterin (6-MI),4-amino-6-methylisoxanthopterin (6-MAP), and 4-dimethylaminopyridine(DMAP); the nucleic acid probes for various genetic applications, suchas the oligonucleotide-conjugated with a fluorescent reporter dye(ALEXA, FAM, TET, TAMRA, CY3, CY5, VIC, JOE, HEX, NED, PET, ROX, TexasRed and others) and/or a fluorescent quenchers (BHQs); the molecularbeacons (MBs), which are single-stranded nucleic acid probes containinga stem-loop structure and a dual fluorophore-and-quencher label; and thenucleic acid aptamers.

Generally, as used herein, a “template” is a polynucleotide, or apolynucleotide mimic, that contains the desired or unknown targetnucleotide sequence. In some instances, the terms “target sequence”,“template polynucleotide”, “target nucleic acid”, “targetpolynucleotide”, “nucleic acid template”, “template sequence, andvariations thereof, are used interchangeably. Specifically, the term“template” refers to a strand of nucleic acid from which a complimentarycopy is synthesized from nucleotides or nucleotide analogues through thereplication by a template-dependent or template-directed nucleic acidpolymerase. Within a nucleic acid duplex, the template strand is, by theconvention definition, depicted and described as the “bottom” strand.Similarly, the non-template strand is often depicted and described asthe “top” strand. The “template” strand may also be referred to as the“sense”, or “plus”, strand and the non-template strand as the“antisense”, or “minus”, strand.

The term “initiator” refers to a mononucleoside, a mononucleotide, andoligonucleotide, a polynucleotide, or modified analogues thereof, fromwhich a nucleic acid is to be synthesized by nucleic acid polymerase denovo. The term “initiator” may also refer to a xeno nucleic acids (XNA)or a peptide nucleic acid (PNA) having a 3′-hydroxyl group.

The terms “nucleotide incorporation”, “analogue incorporation”,“incorporating nucleotide” and “incorporating analogue” are known tothose skilled in the art and used to describe a process or reaction fornucleic acid synthesis. Thus, as used herein, the term “incorporation”is known to flexibly refer to add one, or more nucleotides, or anyspecified nucleic acid precursors to the 3′-hydroxyl terminus of anucleic acid initiator or a primer.

The term “nucleotide analogue” is known to those skilled in the art todescribe the chemically modified nucleotides or artificial nucleotides,which are structural mimics of canonical nucleotides. These nucleotideanalogues can serve as substrates for nucleic acid polymerases tosynthesize nucleic acid. A nucleotide analogue may have one or morealtered components of a nucleotide (e.g., the phosphate backbone,pentose sugar, and nucleobase), which changes the structure andconfiguration of a nucleotide and affects its interactions with othernucleobases and the nucleic acid polymerases. For example, a nucleotideanalogue having altered nucleobase may confer alternative base-pairingand base-stacking properties in the DNA or RNA. Furthermore, by way ofexample, the modification at the base may generate various nucleosidessuch as inosine, methyl-5-deoxycytidine, deoxyuridine,dimethylamino-5-deoxyuridine, diamino-2,6-purine orbromo-5-deoxyuridine, and any other analogues which permitshybridization. In other exemplary aspects, modifications may take placeat the level of sugar moiety (for example, replacement of a deoxyriboseby an analogue), and/or at the level of the phosphate group (forexample, boronate, alkylphosphonate, or phosphorothioate derivatives). Anucleotide analogue monomer may have a phosphate group selected from amonophosphate, a diphosphate, a triphosphate, a tetraphosphate, apentaphosphate, and a hexaphosphate.

Other examples of nucleotide analogues also include nucleotides having aremovable blocking moiety. Examples of the removable blocking moietyinclude, but are not limited to, a 3′-O-blocking moiety, a base blockingmoiety, and a combination thereof. Examples of the 3′-O-blocking moietyinclude, but are not limited to, O-azido (O-N₃), O-azidomethyl, O-amino,O-allyl, O-phenoxyacetyl, O-methoxyacetyl, O-acetyl,O-(p-toluene)sulfonate, O-phosphate, O-nitrate,O-[4-methoxy]-tetrahydrothiopyranyl, O-tetrahydrothiopyranyl,O-[5-methyl]-tetrahydrofuranyl, O-[2-methyl,4-methoxy]-tetrahydropyranyl, O-[5-methyl]-tetrahydropyranyl, andO-tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methyl, and O-acyl groups.Examples of the base blocking moiety may be a reversible dye-terminator.Examples of the reversible dye-terminator include, but are not limitedto, a reversible dye-terminator of Illumina MiSeq, a reversibledye-terminator of Illumina HiSeq, a reversible dye-terminator ofIllumina Genome Analyzer IIX, a reversible dye-terminator of HelicosBiosciences Heliscope, and a reversible dye-terminator of LaserGen’sLightning Terminators.

As used herein, “B-family DNA polymerases (PolBs)” refers to the mostcommon template-dependent DNA polymerases or replicases in all domainsof life and many DNA viruses. Like most nucleic acid polymerases,natural PolBs require a duplex primer-template DNA with a free3′-hydroxyl (3′-OH) group at the primer terminus, all four nucleosidetriphosphates (dATP, dTTP, dCTP, and dGTP), and catalytic divalentcations (Mg²⁺ or Mn²⁺, etc.) for catalyzing the nucleotidyl transferasereaction of adding nucleotides to the 3′-OH terminus of a primer. ThePolB enzymes, such as bacterial Pol II and archaeal B-family DNApolymerases, are replicative and repair polymerases that inherentlycontain a catalytic polymerase domain and a 3′ to 5′ exonucleolytic, orproofreading, domain for removing the mis-incorporated nucleotide fromthe growing primer strand during nucleic acid replication. The term “3′to 5′ exonucleolytic domain” (Exo domain) refers to a region of theamino acid sequence of a polymerase, which exerts the nucleic aciddegradation activity from the 3′-terminus of the primer or thepolynucleotide chain. Coordinately, the term “catalytic polymerasedomain” (Pol domain) refers to a region of the amino acid sequence of apolymerase, which exerts the catalytic DNA/RNA polymerase activity foradding nucleotides to the 3′-terminus of a primer or a polynucleotidechain.

All known structures of PolB catalytic polymerase domain resemble theshape of human right hand, where the key functional regions arecharacterized as fingers, palm, and thumb subdomains. The most conservedregion is the palm subdomain, which contains the essential residues forcatalysis. The protein sequence-alignment among various B-family DNApolymerases from different kingdoms of life and DNA viruses reveals thatthe PolB polymerases generally harbor six semi-conserved or conservedmotifs (I-VI) for their essential exonuclease and polymerase functions.The first three sequence-motifs (Exo I, Exo II, Exo III) are in the Exodomain, while the other three motifs (designated as Motif A, B, and C,respectively) reside in the Pol domain (Hopfner et al, Proc. Natl. Acad.Sci. USA 96, 3600-3605, 1999). In some embodiments of the presentinvention, without being limited by any theory, it is discovered thatthrough modifying some novel positions/regions in said motifs ofB-family DNA polymerases, the polymerases can thereby effectivelycatalyze de novo RNA synthesis as a template-independent RNApolymerases. Based on the acquired activities of de novo RNA synthesis,these modified B-family DNA polymerases are referred to astemplate-independent RNA polymerase variants. In other words, the RNApolymerase variant is capable of incorporating ribonucleotides, such asrATP, rUTP, rCTP and rGTP, at the 3′-terminus of an initiatoroligonucleotide under impeding reaction conditions.

As used herein, the term “mutant” in the context of the presentinvention, means a polypeptide, typically recombinant, that comprisesone or more amino acid substitutions relative to a corresponding,functional DNA polymerase.

As used herein, in the context of the present invention, “correspondingto another sequence” (e.g., regions, fragments, nucleotide or amino acidpositions, or the like) is based on the convention of numberingaccording to nucleotide or amino acid position number and then aligningthe sequences in a manner that maximizes the percentage of sequenceidentity. An amino acid “corresponding to position X of specificsequence” refers to an amino acid in a polypeptide of interest thataligns with the equivalent amino acid of a specified sequence.Generally, as described herein, the amino acid corresponding to aposition of a polymerase can be determined using an alignment algorithmsuch as BLAST and other currently available tools for conducting aminoacid sequence alignment. Because not all positions within a given“corresponding region” need to be identical, non-matching positionswithin a corresponding region may be regarded or define as“corresponding positions”. Accordingly, as used herein, referral to an“amino acid position corresponding to amino acid position X of aspecified DNA polymerase” refers to equivalent positions, based onalignment, in other DNA polymerases and structural homologues andfamilies.

As used herein, the term “consensus sequence of SEQ ID NO: 1” usedherein refers to a reference sequence comprising the conserved orsemi-conserved amino acids of cross-species B-family DNA polymerase. Theconsensus sequence of SEQ ID NO: 1 is a virtual sequence and isgenerated by aligned the following 16 wild-type B-family DNA polymerasesto obtain the conserved amino acids: Thermococcus gorgonarius DNApolymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1),Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcusfuriosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase(Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcinaacetivorans DNA polymerase (Mac), human DNA polymerase delta catalyticp125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase deltacatalytic subunit (ScePOLD), Pyrobaculum islandicum DNA polymerase(Pis), Sulfolobus solfataricus DNA polymerase (Sso), Pseudomonasaeruginosa DNA polymerase II (Pae), Escherichia. coli DNA polymerase II(Eco), Escherichia coli phage RB69 DNA polymerase (RB69), Escherichiacoli phage T4 DNA polymerase (T4), or Bacillus phage Phi29 DNApolymerase (Phi29). These PolB sequences are aligned for obtaining thealignment sequence as a reference of functionally equivalent positions.

The positions of motifs Exo I, Exo II, Exo III, A, B, and C are definedby the inventor using the consensus sequence of SEQ ID NO: 1 of thepresent invention; therefore, it shall be noted that the positions ofthese motifs defined in the present invention are not totally the sameas those described in the literature or prior art.

Objectives

The inventor has discovered PolB variants that have an improved functionand activity for utilizing canonical nucleotides, nucleotide analogues,and initiators for synthesizing polynucleotides in atemplate-independent manner. These PolB variants can efficiently addsaid canonical nucleotides or nucleotide analogues to said initiator inthe absence of a replicative template to synthesize a polynucleotidewith a random or defined sequence.

More specifically, the inventor has discovered PolB variants canefficiently catalyze the additions of natural ribonucleotides (rNTP),such as rATP, rUTP, rCTP and rGTP, to the 3′-OH ends of asingle-stranded nucleic acid initiator or a blunt-end duplex nucleicacid initiator, in the absence of replicative template, to generatepolynucleotides with desired nucleic acid sequences. Furthermore, thePolB variants provided herein generally have a broader substratespecificity, which means the PolB variants can utilize not onlynaturally occurring nucleotides, but also varieties of modifiednucleotides and nucleic acid analogues for the de novo nucleic acidsynthesis. Thus, the modified nucleotide can be further designed forbeing incorporated to the initiator to generate certain functionalpolynucleotides. Therefore, these PolB variants broaden the scope andutility of template-independent enzymatic nucleic acid synthesisapplications for synthesizing polynucleotides with desired sequences andfeatures.

Protein Sequence Alignment OF B-Family DNA Polymerases

FIG. 1 shows the amino acid sequence alignment of 16 different wild-typeB-family DNA polymerases (PolBs) utilized by the inventor, and theoutcome of consensus sequence alignment is listed in the bottom (SEQ IDNO:1). The 16 wild-type PolBs used for alignment are Thermococcusgorgonarius DNA polymerase (Tgo, SEQ ID NO:2), Thermococcus kodakarensisDNA polymerase (Kod1, SEQ ID NO:3), Thermococcus sp. (strain 9°N-7) DNApolymerase (9°N, SEQ ID NO:4), Pyrococcus furiosus DNA polymerase (Pfu,SEQ ID NO:5), Thermococcus litoralis DNA polymerase (Vent, SEQ ID NO:6),Methanosarcina acetivorans DNA polymerase (Mac, SEQ ID NO:7),Pyrobaculum islandicum DNA polymerase, (Pis, SEQ ID NO:8), Sulfolobussolfataricus DNA polymerase (Sso, SEQ ID NO:9), Methanococcusmaripaludis DNA polymerase (Mma, SEQ ID NO: 10), human DNA polymerasedelta catalytic p125 subunit (hPOLD, SEQ ID NO: 11), Saccharomycescerevisiae DNA polymerase delta catalytic subunit (ScePOLD, SEQ ID NO:12), Pseudomonas aeruginosa DNA polymerase II (Pae, SEQ ID NO: 13),Escherichia. coli DNA polymerase II (Eco, SEQ ID NO: 14), Escherichiacoli phage RB69 DNA polymerase (RB69, SEQ ID NO: 15), Escherichia coliphage T4 DNA polymerase (T4, SEQ ID NO: 16), and Bacillus phage Phi29DNA polymerase (Phi29, SEQ ID NO: 17).

As shown in FIG. 1 , various sequence regions among these exemplarywild-type PolBs are highly conserved while other regions are morevariable. Those of skill in the art will immediately recognize andunderstand that mutations in addition to those specifically identifiedand discussed herein may be also made in the variable regions ofwild-type PolBs without altering, or without substantially altering, thepolymerase activity of the mutated enzyme. Likewise, conservativemutations at conserved residues/positions of any of PolBs may be madewithout altering, or substantially altering, the polymerase activity ofthe mutated enzyme. Enzyme engineering based on comparative structureanalysis with other functionally related enzymes or homologs is a usefultechnique in the molecular biology field that allows the inventor toreasonably predict the effect of a given mutation on the catalyticactivity of the enzyme. Based on the present disclosure, using thesequence, structural data, and known physical properties of amino acids,those of skill in the art can mutate enzymes, such as the DNApolymerases encompassed by the present invention, without altering, orwithout substantially altering, the essential, intrinsic characteristicsof the enzymes.

Besides, the motifs Exo I, Exo II, Exo III, A, B, and C corresponding tothe positions 349 to 364, 450 to 476, 590 to 608, 706 to 730, 843 to855, and 940 to 956 respectively, of the consensus sequence of SEQ IDNO:1 are focused in the present disclosure. More specifically, thepolymerase variant in the present invention is based on substitutionmutations at one or more residues correspondingly residing in saidmotifs.

RNA Polymerase Variant

In view of the above, provided herein are altered polymerase, which isdescribed based on the amino acid sequence of the consensus sequence ofSEQ ID NO: 1. An altered polymerase includes substitution mutations atone or more residues when compared to the consensus sequence of SEQ IDNO: 1. A substitution mutation can be at the same, a homologous, or afunctionally equivalent position as compared to the consensus sequenceof SEQ ID NO: 1. The skilled person can readily appreciate that analtered polymerase described herein is not naturally occurring.Therefore, an altered polymerase described herein is based on theconsensus sequence of SEQ ID NO: 1 and further includes substitutionmutations at one or more residues of the corresponding wild-typepolymerase (individual parent polymerase). In one embodiment, at leastone substitution mutation is at a position functionally equivalent to anamino acid of the consensus sequence of SEQ ID NO: 1. “Functionallyequivalent” means that the altered polymerase has the amino acidsubstitution at the amino acid position according to the consensussequence of SEQ ID NO: 1 that has the same functional or structural rolein both the consensus sequence and the altered polymerase.

In general, functionally equivalent substitution mutations in two ormore different polymerases occur at homologous amino acid positions inthe amino acid sequences of the polymerases. Hence, “functionallyequivalent” also encompasses mutations that are “positionallyequivalent” or “homologous” to a given mutation, regardless of whetheror not the particular function of the mutated amino acid is known. It ispossible to identify the regions of functionally equivalent andpositionally equivalent amino acid residues in the amino acid sequencesof two or more different polymerases on the basis of sequence alignmentand/or molecular modelling. For instance, the amino acid sequencealignment of exemplary 16 wild-type B-family DNA polymerases fromdifferent domains of life are used to identify positionally equivalentand/or functionally equivalent residues. The result of the proteinsequence alignment among these PolBs is set forth in FIG. 1 . Thus, forthe exemplary residue 141 of the Tgo, Kod1, 9°N, Pfu, and Ventpolymerases, residue 171 of the Pis, residue 231 of the Sso, and residue198 of the Mac polymerase are functionally equivalent and positionallyequivalent. Likewise, for the exemplary residue 143 of the Tgo, Kod1,9°N, Pfu, and Vent polymerases, residue 173 of the Pis, residue 233 ofthe Sso, and residue 200 of the Mac polymerase are functionallyequivalent and positionally equivalent. The skilled person can easilyidentify functionally equivalent residues in different polymerases.

In accordance with some embodiments, the provided RNA polymerase variantcomprising: a motif A, and a motif B corresponding to positions 706 to730, and 843 to 855, respectively, of a consensus sequence (SEQ ID NO:1); and at least one amino acid substitution (one or more amino acidsubstitutions, or a combination of amino acid substitutions) at aposition in the motif A, the motif B, or the combination thereof;wherein the RNA polymerase variant has reduced or deficient in the 3′ to5′ exonuclease activity. Said the 3′ to 5′ exonuclease activitydeficiency can be reached by any means. For example, practically, the 3′to 5′ exonuclease activity can be reduced, attenuated, removed orinactivated by modifying the 3′ to 5′ exonucleolytic domain of thepolymerase to generate a polymerase that has reduced or is deficient inthe 3′ to 5′ exonuclease activity. Preferably, the means of amino acidsubstitution is adapted to modify the 3′ to 5′ exonucleolytic domain. Inother words, the provided RNA polymerase variant may comprise at leastone amino acid substitution in Exo I, Exo II, Exo III or thecombination. For example, the PolB variants may have functionallyequivalent or positionally equivalent substitutions of the native D withA at position 354 (D354A) and the native E with A at position 356(E356A) in the motif Exo I of SEQ ID NO:1, thereby causing a 3′-5′exonuclease deficiency.

In accordance with some embodiments, the RNA polymerase variant ismodified from a wild-type B-family DNA polymerase having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17., which are respectivelyderived from the wild-type B-family DNA polymerase of Thermococcusgorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNApolymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N),Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNApolymerase (Vent), Methanosarcina acetivorans DNA polymerase (Mac),Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNApolymerase (Sso), Methanococcus maripaludis DNA polymerase (Mma), humanDNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomycescerevisiae DNA polymerase delta catalytic subunit (SecPOLD), Pseudomonasaeruginosa DNA polymerase II (Pae), Escherichia. coli DNA polymerase II(Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phageT4 DNA polymerase (T4), and Bacillus phage Phi29 DNA polymerase (Phi29).

In accordance with certain embodiments, the representative RNApolymerase variant comprises a motif Exo I corresponding to positions349 to 364 of the consensus sequence (SEQ ID NO:1), and the RNApolymerase variant has at least one amino acid substitution at aposition in the motif Exo I. Preferably, an amino acid L or Mcorresponding to position 715 of SEQ ID NO: 1 is substituted with A, C,D, F, G, H, K, N, Q, S, W, or Y; an amino acid Y corresponding toposition 716 of SEQ ID NO: 1 remains unchanged or is substituted with A,C, D, G, N, S, T or V; and an amino acid P corresponding to position 717of SEQ ID NO: 1 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus gorgonarius DNA polymerase (Tgo)having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein: anamino acid L at position 408 of SEQ ID NO: 2 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 409 of SEQ ID NO: 2 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; and an amino acid P at position 410of SEQ ID NO: 2 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus gorgonarius DNA polymerase (Tgo)having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein: anamino acid L at position 408 of SEQ ID NO: 2 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 409 of SEQ ID NO: 2 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; an amino acid P at position 410 ofSEQ ID NO: 2 remains unchanged or is substituted with A, C, G, I, L, M,N, S, T or V, preferably G; and an amino acid A at position 485 of SEQID NO: 2 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus kodakarensis DNA polymerase (Kod1)having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein: anamino acid L at position 408 of SEQ ID NO: 3 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 409 of SEQ ID NO: 3 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; and an amino acid P at position 410of SEQ ID NO: 3 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus kodakarensis DNA polymerase (Kod1)having a wild-type amino acid sequence of SEQ ID NO: 3; and wherein: anamino acid L at position 408 of SEQ ID NO: 3 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 409 of SEQ ID NO: 3 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; an amino acid P at position 410 ofSEQ ID NO: 3 remains unchanged or is substituted with A, C, G, I, L, M,N, S, T or V, preferably G; and an amino acid A at position 485 of SEQID NO: 3 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase(9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; andwherein: an amino acid L at position 408 of SEQ ID NO: 4 is substitutedwith A, C, D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an aminoacid Y at position 409 of SEQ ID NO: 4 remains unchanged or issubstituted with A, C, D, G, N, S, T or V, preferably A; and an aminoacid P at position 410 of SEQ ID NO: 4 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase(9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; andwherein: an amino acid L at position 408 of SEQ ID NO: 4 is substitutedwith A, C, D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an aminoacid Y at position 409 of SEQ ID NO: 4 remains unchanged or issubstituted with A, C, D, G, N, S, T or V, preferably A; an amino acid Pat position 410 of SEQ ID NO: 4 remains unchanged or is substituted withA, C, G, I, L, M, N, S, T or V, preferably G; and an amino acid A atposition 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Pyrococcus furiosus DNA polymerase (Pfu) havinga wild-type amino acid sequence of SEQ ID NO: 5; and wherein: an aminoacid L at position 409 of SEQ ID NO: 5 is substituted with A, C, D, F,G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y at position410 of SEQ ID NO: 5 remains unchanged or is substituted with A, C, D, G,N, S, T or V, preferably A; and an amino acid P at position 411 of SEQID NO: 5 remains unchanged or is substituted with A, C, G, I, L, M, N,S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Pyrococcus furiosus DNA polymerase (Pfu) havinga wild-type amino acid sequence of SEQ ID NO: 5; and wherein: an aminoacid L at position 409 of SEQ ID NO: 5 is substituted with A, C, D, F,G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y at position410 of SEQ ID NO: 5 remains unchanged or is substituted with A, C, D, G,N, S, T or V, preferably A; an amino acid P at position 411 of SEQ IDNO: 5 remains unchanged or is substituted with A, C, G, I, L, M, N, S, Tor V, preferably G; and an amino acid A at position 486 of SEQ ID NO: 5is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W orY, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus litoralis DNA polymerase (Vent)having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein: anamino acid L at position 411 of SEQ ID NO: 6 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 412 of SEQ ID NO: 6 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; and an amino acid P at position 413of SEQ ID NO: 6 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Thermococcus litoralis DNA polymerase (Vent)having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein: anamino acid L at position 411 of SEQ ID NO: 6 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 412 of SEQ ID NO: 6 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; an amino acid P at position 413 ofSEQ ID NO: 6 remains unchanged or is substituted with A, C, G, I, L, M,N, S, T or V, preferably G; and an amino acid A at position 488 of SEQID NO: 6 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Methanosarcina acetivorans DNA polymerase (Mac)having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein: anamino acid L at position 485 of SEQ ID NO: 7 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 486 of SEQ ID NO: 7 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; and an amino acid P at position 487of SEQ ID NO: 7 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Methanosarcina acetivorans DNA polymerase (Mac)having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein: anamino acid L at position 485 of SEQ ID NO: 7 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 486 of SEQ ID NO: 7 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; an amino acid P at position 487 ofSEQ ID NO: 7 remains unchanged or is substituted with A, C, G, I, L, M,N, S, T or V, preferably G; and an amino acid A at position 565 of SEQID NO: 7 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Pyrobaculum islandicum DNA polymerase (Pis)having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein: anamino acid M at position 426 of SEQ ID NO: 8 is substituted with A, C,D, F, G, H, K, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 427 of SEQ ID NO: 8 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; and an amino acid P at position 428of SEQ ID NO: 8 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Pyrobaculum islandicum DNA polymerase (Pis)having a wild-type amino acid sequence of SEQ ID NO: 8; and wherein: anamino acid M at position 426 of SEQ ID NO: 8 is substituted with A, C,D, F, G, H, K, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 427 of SEQ ID NO: 8 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; an amino acid P at position 428 ofSEQ ID NO: 8 remains unchanged or is substituted with A, C, G, I, L, M,N, S, T or V, preferably G; and an amino acid A at position 508 of SEQID NO: 8 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Sulfolobus solfataricus DNA polymerase (Sso)having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein: anamino acid L at position 518 of SEQ ID NO: 9 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 519 of SEQ ID NO: 9 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; and an amino acid P at position 520of SEQ ID NO: 9 remains unchanged or is substituted with A, C, G, I, L,M, N, S, T or V, preferably G.

In accordance with some embodiments, the representative RNA polymerasevariant is derived from Sulfolobus solfataricus DNA polymerase (Sso)having a wild-type amino acid sequence of SEQ ID NO: 9; and wherein: anamino acid L at position 518 of SEQ ID NO: 9 is substituted with A, C,D, F, G, H, K, M, N, Q, S, W, or Y, preferably Y; an amino acid Y atposition 519 of SEQ ID NO: 9 remains unchanged or is substituted with A,C, D, G, N, S, T or V, preferably A; an amino acid P at position 520 ofSEQ ID NO: 9 remains unchanged or is substituted with A, C, G, I, L, M,N, S, T or V, preferably G; and an amino acid A at position 601 of SEQID NO: 9 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,T, V, W or Y, preferably E or L.

In accordance with some embodiments, the representative RNA polymerasevariant exhibits an activity of synthesizing nucleic acids in atemplate-independent manner by adding at least one nucleotide selectedfrom the group of naturally occurring nucleotide, nucleotide analogue,or a mixture thereof, to an extendible initiator.

In certain embodiments, the extendible initiator comprises asingle-stranded oligonucleotide initiator, a blunt ended double-strandedoligonucleotide initiator, or a mixture thereof. In certain embodiments,the extendible initiator is a free form nucleic acid and can be reactedin a liquid phase.

In certain embodiments, the extendible initiator is immobilized on asolid support, wherein the solid support comprises a particle, bead,slide, array surface, membrane, flow cell, well, microwell, nano-well,chamber, microfluidic chamber, channel, microfluidic channel, or anyother surfaces.

In certain embodiments, the at least one nucleotide comprises a ribose.Furthermore, the at least one nucleotide is linked with a detectablelabel, such as fluorophores, enzymes, radioactive phosphates,digoxygenin, or biotin.

In accordance with some embodiments, the representative RNA polymerasevariant exhibits the template-independent nucleic acid synthesisactivity at reaction temperatures ranging from 10° C. to 100° C. Forexample, the reaction temperature is between 10° C. and 20° C., between20° C. and 30° C., between 30° C. and 40° C., between 40° C. and 50° C.,between 50° C. and 60° C., between 60° C. and 70° C., between 70° C. and80° C., between 80° C. and 90° C., between 90° C. and 95° C., between95° C. and 100° C., or any reaction temperatures within a range definedby an upper limit of 15° C., 20° C., 25° C., 30° C., 35° C., 37° C., 40°C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85°C., 90° C., 95° C., or 100° C. and an lower limit of 10° C., 15° C., 20°C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60°C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95° C.

Creation of Polymerase Variants

Various types of mutagenesis techniques are optionally used in thepresent disclosure, e.g., to modify polymerases to create the variantsof the subject application, or using random or semi-random mutationalapproaches. In general, any available mutagenesis procedure can be usedfor making polymerase mutants. Such mutagenesis procedures optionallyinclude selection of altered nucleic acids and polypeptides for one ormore activity of interest. Procedures that can be used include, but arenot limited to: the site-directed point mutagenesis, random pointmutagenesis, in vitro or in vivo homologous recombination (DNA shufflingand combinatorial overlap PCR), mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA, point mismatch repair, mutagenesis using repair-deficienthost strains, restriction-selection and restriction-purification,deletion mutagenesis, mutagenesis by total gene synthesis, degeneratePCR, double-strand break repair, and many others known to skilledperson.

Kit for Performing Template-Independent Nucleic Acid Syntheis Reaction

The present invention also provides a kit that includes the RNApolymerase variant described herein, for performing de novo enzymaticnucleic acid synthesis reaction, comprising: an RNA polymerase variantas described above, wherein the RNA polymerase variant exhibits activityof synthesizing nucleic acids in a template-independent manner by addingat least one nucleotide selected from the group of naturally occurringnucleotide, nucleotide analogue, or a mixture thereof, to an extendibleinitiator, thereby synthesizing a desired nucleic acid sequence.

Optionally, other reagents such as buffers and solutions required forthe RNA polymerase variant and nucleotide solution are also included.Instructions for use of the assembled or packaged components are alsotypically but not necessarily included.

Methods and Uses of RNA Polymerase Variant

In some embodiments, the RNA polymerase variants described herein can beused to add natural ribonucleotides (rNTP), such as rATP, rUTP, rCTP andrGTP, to the 3′-hydroxyl (3′-OH) terminus of a single-stranded or ablunt-end, duplex nucleic acids initiator in a template-independentsynthesis manner to produce polynucleotides with desired sequences.

In some embodiments, the RNA polymerase variants described herein can beused to add natural ribonucleotides (rNTP), such as rATP, rUTP, rCTP andrGTP, to the 3′-OH termini of arrays of clustered single-stranded or ablunt-end, duplex nucleic acids initiators, which are immobilized orphysically confined, and separated on a solid support as describedpreviously; and preferably, the solid support is made of glass andimplemented in the form of silicon wafer. Thus, a multiplexing, parallelde novo nucleic acid synthesis can be performed to synthesize largenumbers of various polynucleotides or nucleic acids with distinctsequences.

In certain embodiments, the RNA polymerase variants described herein canbe used to incorporate the nucleotide conjugates (one of the types ofnucleotide analogue defined previously) covalently linked with anenzyme, an antibody, a chemical group, such as a biotin, adesthiobiotin, or a fluorophore on the base, phosphate moiety, orpentose sugar of nucleotide, to the 3′-terminus of the nucleic acidinitiator in a template-independent synthesis manner.

The incorporation of these nucleotide analogues into the nucleic acidsby RNA polymerase variants during the nucleic acid synthesisconcurrently add the desired component, such as an associated enzyme, anantibody, or a chemical group to the newly synthesized nucleic acids ina base-specific, site-specific, or sequence-specific manner. Commoncomponents used to label or generate nucleic acid probes and conjugatesare known in the art, which include, but are not limited to,radiolabeled nucleotides and nucleotide analogues, modified linkers,such as a biotin, a thiol, an azido, or an amine group, fluorophores,enzymes, and antibodies.

Alternatively, in other embodiments, to label or generate nucleic acidprobes, the post-synthetic modifications of nucleic acids can beachieved by covalently or non-covalently coupling with an enzyme, anantibody, a chemical group, or a fluorophore via a modified linker onthe base, the phosphate moiety, or the pentose sugar of synthesisnucleotide. As a result, the desired component can be covalently ornon-covalently associated with the specific base or connected to the 5′-or 3′-terminus of newly synthesized nucleic acids.

In some embodiments, the RNA polymerase variant-dependent incorporationof linker-modified nucleotide analogues may be used to facilitate thenewly synthesized polynucleotides or nucleic acids to be attached,immobilized or physically confined on various solid surfaces.Retrospectively, in other embodiments, the newly synthesizedsequence-specific nucleic acids with unique labels, tags, orfluorophores can be used in various nucleic acid-based moleculardetections, which include, but are not limited to, the fluorescence insitu hybridization (FISH), TaqMan real-time PCR (RT-PCR), real-timefluorescence ligase chain reaction (RT-LCR), real-time fluorescencerecombinase-polymerase amplification (RPA) assay, and real-timefluorescence loop-mediated isothermal amplification assay.

The present disclosure further provides a method fortemplate-independent synthesis of an RNA oligonucleotide, comprising:

-   (a) providing an initiator oligonucleotide,-   (b) providing an RNA polymerase variant; and-   (c) combining the initiator oligonucleotide, the RNA polymerase    variant and one or more nucleotides under conditions sufficient for    the addition of at least one nucleotide to the 3′ end of the    initiator oligonucleotide.

Once the one or more nucleotides are added to the initiatoroligonucleotide, one or more additional nucleotides can be addedsubsequently in order to synthesize a desired RNA oligonucleotide.Therefore, in certain embodiments, the method further comprises addingone or more natural or modified nucleotides to the 3′ end of theresulting RNA oligonucleotide (i.e., the RNA oligonucleotide formed instep (c)) until a desired RNA sequence is obtained. In certainembodiments, the method further comprises: (d) repeating steps (a)-(c)until a desired RNA sequence is obtained.

In certain embodiments, step (c) is carried out in the presence of oneor more additional enzymes. In certain embodiments, step (c) is carriedout in the presence of a mixture of two or more different enzymes. Themixture of enzymes may comprise more than one distinct RNA polymerasevariants (e.g., 2 or 3 RNA polymerase variants).

In certain embodiments, step (c) is carried out in the presence of oneor more additional enzyme (i.e., auxiliary enzyme), such as specificphosphatases in addition to the RNA polymerase variant. In certainembodiments, step (c) is carried out in the presence of a yeastinorganic pyrophosphatase (PPi-ase) in addition to the RNA polymerasevariant.

In certain embodiments, the reaction in step (c) is carried out in thepresence of one or more additional additives. In certain embodiments,step (c) is carried out in the presence of a crowding agent. In certainembodiments, the crowing agent is polyethylene glycol (PEG) or Ficoll.In certain embodiments, the crowding agent is polyethylene glycol (PEG).In certain embodiments, step (c) is carried out in the presence of anRNase inhibitor. In certain embodiments, step (c) is carried out in thepresence of a non- hydrolyzable nucleotide.

EXAMPLES Example 1: Preparation of RNA Polymerase Variants

The gene synthesis approach and mutagenesis technique are adapted tocreate exemplary RNA polymerase variants according to the properties ofconserved/consensus amino acids in the conserved and semi-conservedregions of selective PolBs, which are disclosed herein. For instance,the well-known site-directed mutagenesis approach is conducted to changethe amino acid residues in the motif Exo I, motif Exo II, motif Exo III,motif A, motif B, and motif C regions of an exemplary wild-type PolBlisted herein.

In some embodiments, the procedure for obtaining exemplary RNApolymerase variants is generally divided into three steps, includingStep 1: Gene synthesis of wild-type PolB and its 3′ to 5′exonuclease-deficient (Exo⁻) mutant, Step 2: Construction of thespecific exemplary RNA polymerase variant in the desired region, andStep 3: Expression and purification of wild-type PolB, Exo⁻ mutant, andRNA polymerase variant. As described in more detail below, thetechniques used in said procedure are well-known to those skilled in theart.

In Step 1, the codon-optimized gene fragment encoding the wild-type,intein-free PolB polymerase is synthesized by Genomics BioSci & Tech Co.(New Taipei City, Taiwan). The 3′ to 5′ exonuclease-deficient(designated as Exo⁻) PolB polymerase is also provided by the samevendor. The superscript “Exo⁻” following an abbreviated name of any PolBlisted herein means that the designated wild-type PolB has been modifiedto eliminate the intrinsic 3′ to 5′ exonuclease activity, whichindicates said PolB is a 3′ to 5′ exonuclease-deficient PolB.Preferably, in the Examples of this disclosure, the Exo⁻ means a PolBmutant carrying combinatory mutations at the positions corresponding toD354 of SEQ ID NO: 1, which is substituted with an alanine residue(D354A), and E356 of SEQ ID NO: 1, which is also substituted with analanine residue (E356A), respectively.

In Step 2, the synthetic wild-type and Exo⁻ PolB genes are respectivelysubcloned into the pET28b vector using the flanking NdeI and NotIrestriction sites. The sequences of recombinant plasmids are confirmedby DNA sequencing. To create the RNA polymerase variant at the desiredmotif region of the PolB Exo⁻ protein backbone, the site-directedmutagenesis, is conducted. Briefly, the site-directed mutagenesis PCR isperformed with the recombinant plasmids using the Q5 Site-directedMutagenesis Kit from New England Biolabs (Ipswich, MA) to introduce theamino acid substitution. The products are first analyzed by 1% agarosegel to confirm the amplicon size and the rest of PCR reaction mixture isthen treated with DpnI at 37° C. for an hour. The mixture is furtherincubated at 70° C. for 10 mins to inactivate the DpnI function. TheDpnI-treated PCR reaction mixture is then purified by the Qiagen’sQIAquick PCR Purification Kit (Whatman, MA). The purified DNA fragmentis treated with the mixture of T4 PNK and T4 DNA ligase. There-circularized PCR-amplified DNA is transformed back into the E. colicells. The plasmid DNA was later extracted from the E. coli cells usingthe Qiagen Plasmid Mini Kit (Whatman, MA). The mutated sequences of thepolymerase variants at the desired motif region, or regions, areconfirmed by DNA sequencing.

In Step 3, E. coli Acella cells harboring the plasmid DNA carryingspecific polymerase variant gene are grown in 2 L of LB mediumsupplemented with 0.5% glucose and 50 µg/ml carbenicillin at 37° C. Whenthe cell density reaches an absorbance value at OD_(600nm) around0.6-0.8, an 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) isadded to induce protein expression. Cells are grown for additional 4hours at 37° C. and then harvested by centrifugation at 4° C. for 10 minat 7,000 ×g. Cell pellets are resuspended with buffer A [50 mM Tris-HCl(pH 7.5), 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v/v) glycerol]containing 1 mM benzamidine hydrochloride. Cell lysis is achieved byincubation with 50 mg of lysozyme on ice for 1 hour followed bysonication. The cell lysate is clarified by centrifugation at 18,000 ×gfor 25 min at 4° C. The clarified crude cell extract is incubated at 70°C. for 30 minutes and then cooled down at 4° C. The heat-treated cellextract is further clarified by centrifugation at 18,000 ×g for 25minutes at 4° C. After centrifugation, the supernatant is diluted withbuffer A without NaCl and loaded onto a HiTrap Heparin column (CytivaLife Sciences, Marlborough, MA, USA) pre-equilibrated in buffer A in theAKTA pure chromatography system (Cytiva Life Sciences, Marlborough, MA,USA). The protein is eluted with the linear 100 mM to 1 M NaCl gradientusing the buffer B [50 mM Tris-HCl (pH 8.0), 1 M NaCl, 0.5 mM EDTA, 1 mMDTT, 5% (v/v) glycerol]. Column fractions are analyzed by 10% SDS-PAGE.Fractions containing desired protein are pooled and dialyzed against thestorage buffer [50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.5 mM EDTA, 1 mMDTT, 5% (v/v) glycerol] at 4° C. overnight. The dialyzed proteinfraction pool containing the target protein is concentrated using anAmicon filter unit (MW cut-off 50,000). The concentrated protein pool isaliquoted and stored at -20° C. Each mutant polymerase variant waspurified with the same procedures as described above. The final proteinconcentration is determined by the Bradford reaction (Bradford, 1976)using the Bio-Rad Protein Assay (Hercules, CA) with bovine serum albuminas a standard.

In this example, selected (exemplary) Exo⁻ RNA polymerase variants aimedfor the following assays with functionally or positionally substitutionsresiding in the motif A and/or motif B are summarized and listed inTable 1

TABLE 1 List of amino acid substitutions in the exemplary RNA polymerasevariants Type of PolB Enzymes SEQ ID NO Equivalent substitutions in themotif A and/or motif B corresponding to the consensus sequence (SEQ IDNO: 1) Position 715 (motif A) Position 716 (motif A) Position 717 (motifA) Position 854 (motif B) Tgo 2 L408A, L408C, L408D, L408F, L408G,L408H, L408K, L408M, L408N, L408Q, L408S, L408W, L408Y Y409A,Y409C,Y409D, Y409G, Y409N, Y409S, Y409T, Y409V P410A,P410C, P410G, P410I,P410L, P410M, P410N, P410S, P410T, P410V A485C,A485D, A485E, A485F,A485G, A485H, A485I, A485K, A485L, A485M, A485N, A485P, A485Q, A485R,A485T, A485V, A485W, A485Y Kod1 3 L408A, L408C, L408D, L408F, L408G,L408H, L408K, L408M, L408N, L408Q, Y409A, Y409C, Y409D, Y409G, Y409N,Y409S, Y409T, Y409V P410A, P410C, P410G, P410I, P410L, P410M, P410N,P410S, P410T, P410V A485C, A485D, A485E, A485F, A485G, A485H, A485I,A485K, A485L, A485M, L408S, L408W, L408Y A485N, A485P, A485Q, A485R,A485T, A485V, A485W, A485Y 9°N 4 L408A, L408C, L408D, L408F, L408G,L408H, L408K, L408M, L408N, L408Q, L408S, L408W, L408Y Y409A, Y409C,Y409D, Y409G, Y409N, Y409S, Y409T, Y409V P410A, P410C, P410G, P410I,P410L, P410M, P410N, P410S, P410T, P410V A485C, A485D, A485E, A485F,A485G, A485H, A485I, A485K, A485L, A485M, A485N, A485P, A485Q, A485R,A485T, A485V, A485W, A485Y Pfu 5 L409A, L409C, L409D, L409F, L409G,L409H, L409K, L409M, L409N, L409Q, L409S, L409W, L409Y Y410A, Y410C,Y410D, Y410G, Y410N, Y410S, Y410T, Y410V P411A, P411C, P411G, P411I,P411L, P411M, P411N, P411S, P411T,P411V A486C, A486D, A486E, A486F,A486G, A486H, A486I, A486K, A486L, A486M, A486N, A486P, A486Q, A486R,A486T, A486V, A486W, A486Y Vent 6 L411A, L,411C, L411D, L,411F, L411G,L,411H, L411K, L411M, L411N, L411Q, L411S, L411W, Y412A, Y412C, Y412D,Y412G, Y412N, Y412S, Y412T, Y412V P413A, P413C, P413G, P413I, P413L,P413M, P413N, P413S, P413T, P413V A488C, A488D, A488E, A488F, A488G,A488H, A488I, A488K, A488L, A488M, A488N, A488P, L411Y A488Q, A488R,A488T, A488V, A488W, A488Y Mac 7 L485A, L485C, L485D, L485F, L485G,L485H, L485K, L485M, L485N, L485Q, L485S, L485W, L485Y Y486A, Y486C,Y486D, Y486G, Y486N, Y486S, Y486T, Y486V P487A, P487C, P487G, P487I,P487L, P487M, P487N, P487S, P487T, P487V A565C, A565D, A565E, A565F,A565G, A565H, A565I, A565K, A565L, A565M, A565N, A565P, A565Q, A565R,A565T, A565V, A565W, A565Y Pis 8 M426A, M426C, M426D, M426F, M426G,M426H, M426K, M426M, M426N, M426Q, M426S, M426W, M426Y Y427A, Y427C,Y427D, Y427G, Y427N, Y427S, Y427T, Y427V P428A, P428C, P428G, P428I,P428L, P428M, P428N, P428S, P428T, P428V A508C, A508D, A508E, A508F,A508G, A508H, A508I, A508K, A508L, A508M, A508N, A508P, A508Q, A508R,A508T, A508V, A508W, A508Y Sso 9 L518A, L518C, L518D, L518F, L518G,L518H, L518K, L518M, L518N, L518Q, Y519A, Y519C, Y519D, Y519G, Y519N,Y519S, Y519T, Y519V P520A, P520C, P520G, P520I, P520L, P520M, P520N,P520S, P520T, P520V A601C, A601D, A601E, A601F, A601G, A601H, A6011,A601K, A601L, A601M, L518S, L518W, L518Y A601N, A601P, A601Q, A601R,A601T, A601V, A601W, A601Y

Example 2: Template-Independent RNA Synthesis Assay

The RNA polymerase variants provided herein are evaluated fortemplate-independent RNA synthesis approach. To further determine theactivities (performance on incorporating naturally occurringribonucleotides) of the RNA polymerase variants, normal rNTPs and asingle-stranded DNA initiator or a blunt-end duplex DNA initiator areused herein. Besides, the reaction temperature of said approach is setup as 55° C. to evaluate the heat-resistance of the exemplary RNApolymerase variants.

In this example, the following synthetic oligonucleotides are used asthe initiators to determine the template-independent RNA synthesisactivity of RNA polymerase variants.

FAMmer ssDNA initiator for Mode I assay: a single-stranded DNA havingthe sequence of

5′-CTCGGCCTGGCACAGGTCCGTTCAGTGCTGCGGCGACCACCGAGG-3′

(SEQ ID NO: 18). This single-stranded oligonucleotide is labeled with afluorescent fluorescein amidite (FAM) dye at the 5′-end.

Blunt-end duplex DNA initiator for Mode II assay: a duplex DNA composedof the FAM-45-mer ssDNA initiator pre-annealed with its complementary45-mer oligonucleotide.

The blunt-end duplex DNA initiator is formed by annealing the FAM-45-merssDNA initiator primer with the complementary 45-mer DNA at a molarratio of 1:1.5 in the 1x TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mMEDTA] containing 100 mM NaCl. The DNA annealing reaction is performed inthe Bio-Rad Thermal Cycler (Hercules, CA) by first heating up the samplemixture to 98° C. for 3 minute and then gradually cooling it down (5°C./30 seconds) to 4° C. The annealing product without overhang is usedas the blunt-end duplex DNA initiator.

The template-independent RNA synthesis reaction is performed in thereaction mixtures (10 µl) containing 100 nM FAM-45-mer ssDNA initiator(Mode I assay) or the blunt-end duplex DNA initiator (Mode II assay),0.25 mM manganese chloride (MnCl₂), and 200 nM exemplary RNA polymerasevariant. The de novo enzymatic RNA synthesis reactions was initiated bythe addition of 100 µM of rNTPs. The reactions were allowed to proceedfor a defined period of time (e.g., 2 minutes for the Mode I assay and10 minutes for the Mode II assay) and then terminated by adding 10 µl of2x quench solution (95% de-ionized formamide and 25 mM EDTA) at aselected reaction temperature (.e.g., 55° C. for either the Mode I orMode II assay). After either the reaction of Mode I or Mode II assay,the sample mixtures were first denatured at 95° C. for 10 min andanalyzed by 20% polyacrylamide gel electrophoresis containing 8 M urea(Urea-PAGE). The de novo enzymatic RNA synthesis reaction products arethen visualized by imaging the gel on the Amersham Typhoon Laser Scanner(Cytiva Life Sciences, Marlborough, MA, USA).

Based on the assays described above, the relative template-independentRNA synthesis activity of each variant is scored and represented by thenumber of symbol “+”. The overall activity score for each variant isdivided into 4 distinct levels: 1) the “+++” indicates that theinitiator is completely elongated to various lengths of newlysynthesized RNA as compared to the band intensity and position of thesubstrate control. Hence, the variant is considered to possess an 100%of RNA synthesis activity; 2) the “++” indicates that the initiator iselongated around 50% to 100% to various lengths of newly synthesized RNAas compared to the band intensity and position of the substrate control.Hence, the variant is considered to possess a 50% to an 100% of RNAsynthesis activity; 3) the “+” indicates that the initiator is elongatedaround 10% to 50% to various lengths of newly synthesized RNA ascompared to the band intensity and position of the substrate control.Hence, the variant is considered to possess a 10% to 50% of RNAsynthesis activity; and 4) the “+/-” indicates that the initiator iselongated less than 10% to various lengths of newly synthesized RNA ascompared to the band intensity and position of the substrate control.Therefore, the variant is considered to possess <10% of RNA synthesisactivity. This principle for activity scoring is applied throughout thepresent disclosure and resulting data are primarily listed in thecorresponding tables.

Example 3: Catalytic Activity of RNA Polymerase Variants onIncorporating rNTPs to the FAM-45-mer ssDNA Initiator and Blunt-EndDuplex DNA Initiator

In this section, the selected exonuclease-deficient RNA polymerasevariants (e.g., Tgo^(exo-), Kod1^(exo-), 9 °N^(exo-), Pfu^(exo-),Vent^(exo-), Mac^(exo-) and Sso^(exo-)) were modified to include on ormore amino acid substitutions with different amino acids in variedconserved regions or motifs of each protein.

Additionally, in the preliminary screening, the inventor has discoveredthat a conserved motif of those exonuclease-deficient RNA polymerasevariants, which is functionally and positionally equivalent to the L715,Y716, and P717 residing in the motif A of the consensus sequence (SEQ IDNO: 1) as defined herein, may be predominantly related with the functionof de novo RNA synthesis. More specifically, said conserved motif may bean essential site for template-independent RNA synthesis activity.Furthermore, a conserved residue residing in the motif B, which isfunctionally and positionally equivalent to the A854 of the consensussequence (SEQ ID NO: 1), serves as a reinforceable site for enhancingthe template-independent RNA synthesis activity (data not shown).

Example 3.1: Template-Independent RNA Synthesis Activity of VentVariants

In this example, the RNA polymerase variants derived from Vent (SEQ IDNO: 6) is used exemplarily for evaluating the template-independent RNAsynthesis activity of the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B. Additionally, the U.S.Pat. No. US11136564B2 disclosed an AAI motif for substituting saidconserved motif of some archaeal DNA polymerases to improve theincorporation of nucleotide analogues for template-dependent DNAsynthesis reactions (i.e., DNA sequencing). The conserved motif AAI isfunctionally and positionally equivalent to the L715, Y716, and P717 ofthe consensus sequence (SEQ ID NO: 1), therefore, the conserved motif isalso functionally and positionally equivalent to the L411, Y412 and P413residing in the motif A of the wild-type Vent (SEQ ID NO: 6). Thus, inview of the effects of the AAI motif on the template-directed nucleotideincorporation, the AAI motif substitution is equivalently included inthis example for comparison. Moreover, in this example, the combinatoryeffect of substitution of A854 residing in the motif B of the consensussequence, which is functionally and positionally equivalent to the A488residing in the motif B of the wild-type Vent, is also evaluated.

In this example, the variants modified from Vent Exo⁻ backbone areevaluated using the Mode I and Mode II activity assays as describedabove. The results of Mode I assay are shown in Table 2.1 and FIG. 2A;and the results of Mode II assay are shown in Table 2.2 and FIG. 2B,where “S” denoted in the figures stands for the substrate (FAM-45-merssDNA initiator or blunt-end duplex DNA initiator) and serves as a blankDNA control. Besides, for the sake of brevity, only the exemplaryresults for the representative Vent variants are shown in FIGS. 2A and2B.

TABLE 2.1 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Vent variants determined by Mode I activityassay Variant Name Substitutions Activity Scoring V01 D141A+E143A - V02D141A + E143A + A488L - V03 D141A + E143A + P413E +/- V04 D141A +E143A + L411Y +/- V05 D141A + E143A + L411A + A488L +/- V06 D141A +E143A + L411C + A488L +/- V07 D141A + E143A + L411D + A488L +/- V08D141A + E143A + L411F + A488L ++ V09 D141A + E143A + L411G + A488L +/-V10 D141A + E143A + L411H + A488L ++ V11 D141A + E143A + L411K + A488L+/- V12 D141A + E143A + L411M + A488L +/- V13 D141A + E143A + L411Q +A488L +/- V14 D141A + E143A + L411Y + A488L ++ V15 D141A + E143A +Y412A + A488L + V16 D141A + E143A + Y412C + A488L +/- V17 D141A +E143A + Y412G + A488L + V18 D141A + E143A + Y412N + A488L +/- V19D141A + E143A + Y412S + A488L + V20 D141A + E143A + P413S + A488L ++ V21D141A + E143A + L411A + Y412A + P413G + A488L ++ V22 D141A + E143A +L411C + Y412A + P413G + A488L +++ V23 D141A + E143A + L411D + Y412A +P413G + A488L + V24 D141A + E143A + L411E + Y412A + P413G + A488L + V25D141A + E143A + L411F +Y412A + P413G + A488L +++ V26 D141A + E143A +L411G + Y412A + P413G + A488L + V27 D141A + E143A + L411H + Y412A +P413G + A488L +++ V28 D141A + E143A + L411I + Y412A+ P413G + A488L + V29D141A + E143A + L411K + Y412A + P413G + A488L +++ V30 D141A + E143A +Y412A + P413G + A488L + V31 D141A + E143A + L411M + Y412A + P413G +A488L +++ V32 D141A + E143A + L411N + Y412A + P413G + A488L ++ V33D141A + E143A + L411Q + Y412A + P413G + A488L +++ V34 D141A + E143A +L411S + Y412A + P413G + A488L ++ V35 D141A + E143A + L411T + Y412A +P413G + A488L + V36 D141A + E143A + L411V + Y412A + P413G + A488L + V37D141A + E143A + L411W + Y412A + P413G + A488L ++ V38 D141A + E143A +L411Y + Y412A + P413G + A488L ++ V39 D141A + E143A + L411Y + Y412C +P413G + A488L + V40 D141A + E143A + L411Y + Y412D + P413G + A488L ++ V41D141A + E143A + L411Y + Y412F + P413G + A488L + V42 D141A + E143A +L411Y + Y412G + P413G + A488L ++ V43 D141A + E143A + L411Y + Y412H +P413G + A488L + V44 D141A+ E143A + L411Y + Y412I + P413G + A488L + V45D141A + E143A + L411Y + Y412L + P413G + A488L + V46 D141A + E143A +L411Y + Y412M + P413G + A488L + V47 D141A + E143A + L411Y + Y412N +P413G + A488L ++ V48 D141A+ E143A + L411Y + Y412Q + P413G + A488L + V49D141A + E143A + L411Y + Y412S + P413G + A488L ++ V50 D141A + E143A +L411Y + Y412T + P413G + A488L ++ V51 D141A + E143A + L411Y + Y412V +P413G + A488L ++ V52 D141A + E143A + L411Y + P413G + A488L ++ V53D141A + E143A + L411Y + Y412A + P413A + A488L ++ V54 D141A + E143A +L411Y + Y412A + P413C + A488L ++ V55 D141A + E143A + L411Y + Y412A +P413D + A488L + V56 D141A + E143A + L411Y + Y412A + P413E + A488L + V57D141A + E143A + L411Y + Y412A + P413F + A488L + V58 D141A + E143A +L411Y + Y412A + P413H + A488L + V59 D141A + E143A + L411Y + Y412A +P413I + A488L ++ V60 D141A + E143A + L411Y +Y412A + P413K + A488L + V61D141A + E143A + L411Y + Y412A + P413L + A488L ++ V62 D141A + E143A +L411Y +Y412A + P413M + A488L ++ V63 D141A + E143A + L411Y + Y412A +P413N + A488L ++ V64 D141A + E143A + L411Y + Y412A + A488L ++ V65D141A + E143A + L411Y + Y412A + P413Q + A488L + V66 D141A + E143A +L411Y + Y412A + P413S + A488L ++ V67 D141A + E143A+ L411Y + Y412A +P413T + A488L ++ V68 D141A + E143A + L411Y + Y412A+ P413V + A488L ++ V69D141A + E143A + L411Y + Y412A + P413W + A488L + V70 D141A + E143A +L411Y + Y412A + P413Y + A488L + V71 D141A + E143A + L411Y + Y412A +P413G + A488A + V72 D141A + E143A + L411Y + Y412A + P413G + A488C ++ V73D141A + E143A+ L411Y +Y412A+ P413G + A488D ++ V74 D141A + E143A +L411Y + Y412A + P413G + A488E ++ V75 D141A + E143A + L411Y + Y412A +P413G + A488F ++ V76 D141A + E143A + L411Y + Y412A + P413G + A488G ++V77 D141A + E143A+ L411Y +Y412A+ P413G + A488H ++ V78 D141A + E143A +L411Y + Y412A + P413G + A488I ++ V79 D141A + E143A + L411Y + Y412A +P413G + A488K ++ V80 D141A + E143A + L411Y + Y412A + P413G + A488M ++V81 D141A + E143A+ L411Y +Y412A+ P413G + A488N ++ V82 D141A + E143A +L411Y + Y412A + P413G + A488P + V83 D141A + E143A + L411Y + Y412A +P413G + A488Q ++ V84 D141A + E143A + L411Y + Y412A + P413G + A488R ++V85 D141A + E143A + L411Y + Y412A + P413G + A488T ++ V86 D141A + E143A +L411Y + Y412A + P413G + A488V ++ V87 D141A + E143A + L411Y + Y412A +P413G + A488W ++ V88 D141A + E143A+ L411Y +Y412A+ P413G + A488Y ++

TABLE 2.2 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Vent variants determined by Mode II activityassay Variant Name Substitutions Activity Scoring V02 D141A + E143A +A488L - V05 D141A + E143A + L411A + A488L +++ V06 D141A + E143A +L411C + A488L +++ V07 D141A + E143A + L411D + A488L +++ V08 D141A +E143A + L411F + A488L +++ V09 D141A + E143A + L411G + A488L -/+ V10D141A + E143A + L411H + A488L ++ V11 D141A + E143A + L411K + A488L -/+V12 D141A + E143A + L411M + A488L ++ V13 D141A + E143A + L411Q + A488L++ V14 D141A + E143A + L411Y + A488L +++ V16 D141A + E143A + Y412C +A488L +++ V17 D141A + E143A + Y412G + A488L +++ V18 D141A + E143A +Y412N + A488L +++ V19 D141A + E143A + Y412S + A488L +++ V20 D141A +E143A + P413S + A488L +++ V38 D141A + E143A + L411Y + Y412A + P413G +A488L +++ V21 D141A + E143A + L411A + Y412A + P413G + A488L +++ V22D141A + E143A + L411C + Y412A + P413G + A488L +++ V23 D141A + E143A +L411D + Y412A + P413G + A488L +++ V24 D141A + E143A + L411E + Y412A +P413G + A488L +++ V25 D141A + E143A + L411F +Y412A + P413G + A488L +++V26 D141A + E143A + L411G + Y412A + P413G + A488L +++ V27 D141A +E143A + L411H + Y412A + P413G + A488L +++ V28 D141A + E143A + L411I +Y412A+ P413G + A488L +++ V29 D141A + E143A + L411K + Y412A + P413G +A488L +++ V30 D141A + E143A + Y412A + P413G + A488L +++ V31 D141A +E143A + L411M + Y412A + P413G + A488L +++ V32 D141A + E143A + L411N +Y412A + P413G + A488L +++ V33 D141A + E143A + L411Q + Y412A + P413G +A488L +++ V34 D141A + E143A + L411S + Y412A + P413G + A488L +++ V35D141A + E143A + L411T + Y412A + P413G + A488L +++ V36 D141A + E143A +L411V + Y412A + P413G + A488L +++ V37 D141A + E143A + L411W + Y412A +P413G + A488L +++ V52 D141A + E143A + L411Y + P413G + A488L +++ V39D141A + E143A + L411Y + Y412C + P413G + A488L +++ V40 D141A + E143A +L411Y + Y412D + P413G + A488L +++ V41 D141A + E143A + L411Y + Y412F +P413G + A488L +++ V43 D141A + E143A + L411Y + Y412H + P413G + A488L +++V44 D141A+ E143A + L411Y + Y412I + P413G + A488L +++ V45 D141A + E143A +L411Y + Y412L + P413G + A488L +++ V46 D141A + E143A + L411Y + Y412M +P413G + A488L +++ V47 D141A + E143A + L411Y + Y412N + P413G + A488L +++V48 D141A + E143A + L411Y + Y412Q + P413G + A488L +++ V49 D141A +E143A + L411Y + Y412S + P413G + A488L +++ V50 D141A + E143A + L411Y+Y412T + P413G + A488L +++ V51 D141A + E143A + L411Y + Y412V + P413G +A488L +++ V64 D141A + E143A + L411Y + Y412A + A488L +++ V53 D141A +E143A + L411Y + Y412A + P413A + A488L +++ V54 D141A + E143A + L411Y +Y412A + P413C + A488L +++ V55 D141A + E143A + L411Y + Y412A + P413D +A488L +++ V56 D141A + E143A + L411Y + Y412A + P413E + A488L +++ V57D141A + E143A + L411Y + Y412A + P413F + A488L +++ V58 D141A + E143A +L411Y + Y412A + P413H + A488L +++ V59 D141A + E143A + L411Y + Y412A +P413I + A488L +++ V60 D141A + E143A + L411Y +Y412A + P413K + A488L +++V61 D141A + E143A + L411Y + Y412A + P413L + A488L +++ V62 D141A +E143A + L411Y + Y412A + P413M + A488L +++ V63 D141A + E143A + L411Y +Y412A + P413N + A488L +++ V65 D141A + E143A + L411Y + Y412A + P413Q +A488L +++ V66 D141A + E143A + L411Y + Y412A + P413S + A488L +++ V67D141A + E143A + L411Y + Y412A + P413T + A488L +++ V68 D141A + E143A +L411Y + Y412A+ P413V + A488L +++ V69 D141A + E143A + L411Y + Y412A +P413W + A488L +++ V70 D141A + E143A + L411Y + Y412A + P413Y + A488L +++V72 D141A + E143A + L411Y + Y412A + P413G + A488C +++ V73 D141A + E143A+L411Y +Y412A+ P413G + A488D +++ V74 D141A + E143A + L411Y + Y412A +P413G + A488E +++ V75 D141A + E143A + L411Y + Y412A + P413G + A488F +++V77 D141A + E143A+ L411Y +Y412A+ P413G + A488H +++ V78 D141A + E143A +L411Y + Y412A + P413G + A488I +++ V79 D141A + E143A + L411Y + Y412A +P413G + A488K +++ V80 D141A + E143A + L411Y + Y412A + P413G + A488M +++V81 D141A + E143A + L411Y + Y412A + P413G + A488N +++ V82 D141A +E143A + L411Y + Y412A + P413G + A488P +++ V83 D141A + E143A + L411Y +Y412A + P413G + A488Q +++ V84 D141A + E143A + L411Y + Y412A + P413G +A488R +++ V85 D141A + E143A + L411Y + Y412A + P413G + A488T +++ V86D141A + E143A + L411Y + Y412A + P413G + A488V +++ V87 D141A + E143A +L411Y + Y412A + P413G + A488W +++ V88 D141A + E143A+ L411Y +Y412A+P413G + A488Y +++

As shown in Table 2.1, Table 2.2, FIG. 2A, and FIG. 2B, the variantscarrying amino acid substitutions in the motif Exo I, motif A and/ormotif B (A488L) have exerted prominent catalytic activity oftemplate-independent enzymatic RNA synthesis in both Mode I and Mode IIassays. More specifically, the preferable substitution combinationsoccurs in the motif A, such as L411A + Y412A + P413G (AAG), L411C +Y412A + P413G (CAG), L411F + Y412A + P413G (FAG), L411H + Y412A + P413G(HAG), L411K + Y412A + P413G (KAG), L411M + Y412A + P413G (MAG), L411Q +Y412A + P413G (QAG), and L411Y + Y412A + P413G (YAG) for the catalyticactivity of template-independent enzymatic RNA synthesis.

Example 3.2: Template-Independent RNA Synthesis Activity of Pfu Variants

In this example, the RNA polymerase variants derived from Pfu (SEQ IDNO: 5) is used exemplarily for evaluating the template-independent RNAsynthesis activity of the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B.

Specifically, the variants modified from Pfu Exo⁻ backbone are evaluatedusing the Mode I and Mode II activity assays as described above. Theresults of Mode I assay are shown in Table 3.1 and FIG. 3A; and theresults of Mode II assay are shown in Table 3.2 and FIG. 3B, where “S”denoted in the figures stands for the substrate (FAM-45-mer ssDNAinitiator or blunt-end duplex DNA initiator) and serves as a blank DNAcontrol. Besides, for the sake of brevity, only the exemplary resultsfor the representative Pfu variants are shown in this example.

TABLE 3.1 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Pfu variants determined by Mode I activityassay Variant Name Substitutions Activity Scoring P01 D141A + E143A -P02 D 141A + E143A + A486L +/ P03 D141A + E143A + L409Y + Y410A+ P411G +P04 D141A + E143A + L409Y + Y410A + P411G + A486L +++ P05 D141A +E143A + L409A + Y410A + P411I + A486L +++

TABLE 3.2 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Pfu variants determined by Mode II activityassay Variant Name Substitutions Activity Scoring P01 D141A + E143A -P02 D141A + E143A + A486L +/ P03 D141A + E143A + L409Y +Y410A + P411G+++ P04 D141A + E143A + L409Y + Y410A + P411G + A486L +++ P05 D141A +E143A + L409A + Y410A + P411I + A486L +++

As shown in FIG. 3A, FIG. 3B, Table 3.1 and Table 3.2, the variantscarrying amino acid substitutions in the motif Exo I and motif A, suchas variants P03, P04, and P05, have exerted prominent catalytic activityof template-independent enzymatic RNA synthesis in both Mode I and ModeII assays. Moreover, the variants carrying combinatory amino acidsubstitutions in the motif Exo I, motif A, and motif B, such as variantsP04 and P05, further enhanced the said catalytic activity.

Example 3.3: Template-Independent RNA Synthesis Activity of Kod1Variants

In this example, the RNA polymerase variants derived from Kod1 (SEQ IDNO: 3) is used exemplarily for evaluating the template-independent RNAsynthesis activity of the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B.

Specifically, the variants modified from Kod1 Exo⁻ backbone areevaluated using the Mode I and Mode II activity assays as describedabove. The results of Mode I assay are shown in Table 4.1 and FIG. 4A;and the results of Mode II assay are shown in Table 4.2 and FIG. 4B,where “S” denoted in the figures stands for the substrate (FAM-45-merssDNA initiator or blunt-end duplex DNA initiator) and serves as a blankDNA control. Besides, for the sake of brevity, only the exemplaryresults for the representative Kod1 variants are shown in this example.

TABLE 4.1 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Kod1 variants determined by Mode I activityassay Variant Name Substitutions Activity Scoring K01 D141A + E143A -K02 D141A + E143A + A485L + K03 D141A + E143A + L408Y + Y409A + P410G ++K04 D141A + E143A + L408Y + Y409A + P410G + A485L +++ K05 D141A +E143A + L408A + Y409A + P410I + A485L ++

TABLE 4.2 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Kod1 variants determined by Mode II activityassay Variant Name Substitutions Activity Scoring K01 D141A + E143A -K02 D141A + E143A + A485L + K03 D141A + E143A + L408Y + Y409A + P410G+++ K04 D141A + E143A + L408Y + Y409A + P410G + A485L +++ K05 D141A +E143A + L408A + Y409A + P410I + A485L +++

As shown in FIG. 4A, FIG. 4B, Table 4.1 and Table 4.2, the variantscarrying amino acid substitutions in the motif Exo I, motif A and/ormotif B, such as variants K02, K03, K04, and K05, have exerted prominentcatalytic activity of template-independent enzymatic RNA synthesis inboth Mode I and Mode II assays. Moreover, the variants carryingcombinatory amino acid substitutions in the motif Exo I, motif A, andmotif B (A485L), such as K04 and K05, further enhanced the saidcatalytic activity.

Example 3.4: Template-Independent RNA Synthesis Activity of Mac Variants

In this example, the RNA polymerase variants derived from Mac (SEQ IDNO: 7) is used exemplarily for evaluating the template-independent RNAsynthesis activity of the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B.

Specifically, the variants modified from Mac Exo⁻ backbone are evaluatedusing the Mode I and Mode II activity assays as described above. Theresults of Mode I assay are shown in Table 5.1 and FIG. 5A; and theresults of Mode II assay are shown in Table 5.2 and FIG. 5B, where “S”denoted in the figures stands for the substrate (FAM-45-mer ssDNAinitiator or blunt-end duplex DNA initiator) and serves as a blank DNAcontrol. Besides, for the sake of brevity, only the exemplary resultsfor the representative Mac variants are shown in this example.

TABLE 5.1 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Mac variants determined by Mode I activityassay Variant Name Substitutions Activity Scoring M01 D198A + E200A -M02 D 198A + E200A + L485Y + Y486A + P487G +/- M03 D 198A + E200A +L485Y + Y486A + P487G + A565L +

TABLE 5.2 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Mac variants determined by Mode II activityassay Variant Name Substitutions Activity Scoring M01 D198A + E200A -M02 D198A + E200A + L485Y + Y486A + P487G +++ M03 D 198A + E200A +L485Y + Y486A + P487G + A565L +++

As shown in FIG. 5A, FIG. 5B, Table 5.1 and Table 5.2, the variantscarrying amino acid substitutions in the motif Exo I and motif A, suchas variant M02, have exerted prominent catalytic activity oftemplate-independent enzymatic RNA synthesis in both Mode I and Mode IIassays. Moreover, the variants carrying combinatory amino acidsubstitutions in the motif Exo I, motif A, and motif B, such as variantM03, further enhanced the said catalytic activity.

Example 3.5: Template-Independent RNA Synthesis Activity of Tgo Variants

In this example, the RNA polymerase variants derived from Tgo (SEQ IDNO: 2) is used exemplarily for evaluating the template-independent RNAsynthesis activity the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B.

Specifically, the variants modified from Tgo Exo⁻ backbone are evaluatedusing the Mode I and Mode II activity assays as described above. Theresults of Mode I assay are shown in Table 6.1 and FIG. 6A; and theresults of Mode II assay are shown in Table 6.2 and FIG. 6B, where “S”denoted in the figures stands for the substrate (FAM-45-mer ssDNAinitiator or blunt-end duplex DNA initiator) and serves as a blank DNAcontrol. Besides, for the sake of brevity, only the exemplary resultsfor the representative Tgo variants are shown in this example.

TABLE 6.1 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Tgo variants determined by Mode I activityassay Variant Name Substitutions Activity Scoring T01 D141A + E143A +L408A + Y409A + P410A + A485L +++ T02 D141A + E143A + L408Y + Y409A +P410G + A485L +++

TABLE 6.2 List of amino acid substitutions and RNA synthesis activityscorings of the exemplary Tgo variants determined by Mode II activityassay Variant Name Substitutions Activity Scoring T01 D141A + E143A +L408A + Y409A + P410A + A485L +++ T02 D141A + E143A + L408Y + Y409A +P410G + A485L +++

As shown in FIG. 6A, FIG. 6B, Table 6.1 and Table 6.2, the variantscarrying amino acid substitutions in motif Exo I, motif A, and motif B,such as variants T01 and T02, have exerted prominent catalytic activityof template-independent enzymatic RNA synthesis in both Mode I and ModeII assays.

Example 3.6: Template-Independent RNA Synthesis Activity of Sso Variants

In this example, the RNA polymerase variants derived from Sso (SEQ IDNO: 9) is used exemplarily for evaluating the template-independent RNAsynthesis activity of the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B.

Specifically, the variants modified from Sso Exo⁻ backbone are evaluatedusing the Mode I and Mode II activity assays as described above. Theresults of Mode I and Mode II assays are shown in Tables 7.1 and 7.2respectively. For the sake of brevity, the Urea-PAGE image results inthis example is currently omitted.

TABLE 7.1 List of amino acid substitutions and activity scorings of theexemplary Sso variants determined by Mode I activity assay Variant NameSubstitutions Activity Scoring S01 D231A + E233A - S03 D231A + E233A +L518Y + Y519A + P520G +++ S06 D231A + E233A + L518Y + Y519A + P520G +A601L +++

TABLE 7.2 List of amino acid substitutions and activity scorings of theexemplary Sso variants determined by Mode II activity assay Variant NameSubstitutions Activity Scoring S01 D231A + E233A - S03 D231A + E233A +L518Y + Y519A + P520G +++ S06 D231A + E233A + L518Y + Y519A + P520G +A601L +++

As shown in the Tables 7.1 and 7.2, the variants carrying amino acidsubstitutions in motif Exo I, motif A, and/or motif B, such as variantsS03 and S06, have exerted prominent catalytic activity oftemplate-independent enzymatic RNA synthesis in both Mode I and Mode IIassays.

Example 3.7: Template-Independent RNA Synthesis Activity of 9°N Variants

In this example, the RNA polymerase variants derived from 9°N (SEQ IDNO: 4) is used exemplarily for evaluating the template-independent RNAsynthesis activity of the variants carrying combinatory substitutions inthe motif Exo I, the motif A, and the motif B. Specifically, thevariants modified from 9°N Exo⁻ backbone are evaluated using the Mode Iand Mode II activity assays as described above. The results of Mode Iand Mode II assays are shown in Tables 8.1 and 8.2 respectively. For thesake of brevity, the Urea-PAGE image results in this example iscurrently omitted.

TABLE 8.1 List of amino acid substitutions and activity scorings of theexemplary 9°N variants determined by Mode I activity assay Variant NameSubstitutions Activity Scoring N02 D141A + E143A + L408Y + Y409A +P410E + A485V ++ N03 D141A + E143A + L408Y + Y409A + P410F + A485V ++N04 D141A + E143A + L408Y + Y409A + P410G + A485V +++ N05 D141A +E143A + L408Y + Y409A + P410H + A485V +++ N06 D141A + E143A + L408Y +Y409A + P410T + A485V +++ N07 D141A + E143A + L408Y + Y409A + P410V +A485V +++ N08 D141A + E143A + L408Y + Y409C + P410G + A485V ++ N09D141A + E143A + L408Y + Y409G + P410G + A485V ++ N10 D141A + E143A +L408Y + Y409I + P410G + A485V ++ N11 D141A + E143A + L408Y + Y409K +P410G + A485V +/ N12 D141A + E143A + L408Y + Y409L + P410G + A485V +/N13 D141A + E143A + L408Y + Y409Q + P410G + A485V +/ N14 D141A + E143A +L408Y + Y409Y + P410G + A485V +++ N15 D141A+ E143A + L408A + Y409A +P410G + A485V ++ N16 D141A + E143A + L408S + Y409A + P410G + A485V ++N17 D141A + E143A + L408V + Y409A + P410G + A485V +++

TABLE 8.2 List of amino acid substitutions and activity scorings of theexemplary 9°N variants determined by Mode II activity assay Variant NameSubstitutions Activity Scoring N02 D141A + E143A + L408Y + Y409A +P410E + A485V +++ N03 D141A + E143A + L408Y + Y409A+ P410F + A485V +++N04 D141A + E143A + L408Y + Y409A + P410G + A485V +++ N05 D141A +E143A + L408Y + Y409A + P410H + A485V +++ N06 D141A + E143A + L408Y +Y409A + P410T + A485V +++ N07 D141A + E143A + L408Y + Y409A + P410V +A485V +++ N08 D141A + E143A + L408Y + Y409C + P410G + A485V +++ N09D141A + E143A + L408Y + Y409G + P410G + A485V +++ N10 D141A + E143A +L408Y + Y409I + P410G + A485V +++ N11 D141A + E143A + L408Y + Y409K +P410G + A485V +++ N12 D141A + E143A + L408Y + Y409L + P410G + A485V +++N13 D141A + E143A + L408Y + Y409Q + P410G + A485V +++ N14 D141A +E143A + L408Y + Y409Y + P410G + A485V +++ N15 D141A + E143A + L408A +Y409A + P410G + A485V +++ N16 D141A + E143A + L408S + Y409A + P410G +A485V +++ N17 D141A+ E143A+ L408V + Y409A + P410G + A485V ++

As shown in the Tables 8.1 and 8.2, the variants carrying amino acidsubstitutions in motif Exo I, motif A, and motif B, such as the variantslisted in Tables 8.1 and 8.2, have exerted prominent catalytic activityof template-independent enzymatic RNA synthesis in both Mode I and ModeII assays.

In view of the results observed, the RNA polymerase variants and the kitprovided herein have been further proven in various scenarios to userNTPs effectively and efficiently for de novo enzymatic RNA synthesis.Furthermore, these RNA polymerase variants are also proven tosuccessfully exert the conferred template-independent RNA synthesisfunction under broader reaction temperatures covering from atmospherictemperatures to the hyperthermal conditions, demonstrating a higherthermotolerance. Therefore, the RNA polymerase variants and the kitwithin the scope of the present disclosure can broaden the scope ofvarious applications of template-independent enzymatic nucleic acidssynthesis in different reaction conditions.

The present disclosure has been described with embodiments thereof, andit is understood that various modifications, without departing from thescope of the present disclosure, are in accordance with the embodimentsof the present disclosure. Hence, the embodiments described are intendedto cover the modifications within the scope of the present disclosure,rather than to limit the present disclosure. The scope of the claimstherefore should be accorded the broadest interpretation so as toencompass all such modifications.

What is claimed is:
 1. An RNA polymerase variant comprising: a motif A,and a motif B corresponding respectively to positions 706 to 730, and843 to 855 of a consensus sequence (SEQ ID NO:1); and at least one aminoacid substitution at a position in the motif A, the motif B, or thecombination thereof; wherein the RNA polymerase variant has a reduced ordeficient 3′ to 5′ exonuclease activity.
 2. The RNA polymerase variantof claim 1, wherein the RNA polymerase variant is modified from awild-type B-family DNA polymerase having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 and
 17. 3. The RNA polymerase variant of claim 2,wherein the wild-type B-family DNA polymerase is Thermococcusgorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNApolymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N),Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNApolymerase (Vent), Methanosarcina acetivorans DNA polymerase (Mac),Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNApolymerase (Sso), Methanococcus maripaludis DNA polymerase (Mma), humanDNA polymerase, delta catalytic p125 subunit (hPOLD), Saccharomycescerevisiae DNA polymerase delta catalytic subunit (ScePOLD), Pseudomonasaeruginosa DNA polymerase II (Pae), Escherichia. coli DNA polymerase II(Eco), Escherichia coli phage RB69 DNA polymerase (RB69), Escherichiacoli phage T4 DNA polymerase (T4), or Bacillus phage Phi29 DNApolymerase (Phi29).
 4. The RNA polymerase variant of claim 1, whereinthe RNA polymerase variant comprises a motif Exo I corresponding topositions 349 to 364 of the consensus sequence (SEQ ID NO:1), and theRNA polymerase variant has at least one amino acid substitution at aposition in the motif Exo I.
 5. The RNA polymerase variant of claim 4,wherein: i. an amino acid L or M corresponding to position 715 of SEQ IDNO: 1 is substituted with A, C, D, F, G, H, K, N, Q, S, W, or Y; ii. anamino acid Y corresponding to position 716 of SEQ ID NO: 1 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; and iii. anamino acid P corresponding to position 717 of SEQ ID NO: 1 remainsunchanged or is substituted with A, C, G, I, L, M, N, S, T or V.
 6. TheRNA polymerase variant according to claim 4, wherein the RNA polymerasevariant is derived from Thermococcus gorgonarius DNA polymerase (Tgo)having a wild-type amino acid sequence of SEQ ID NO: 2; and wherein: i.an amino acid L at position 408 of SEQ ID NO: 2 is substituted with A,C, D, F, G, H, K, M, N, Q, S, W, or Y; ii. an amino acid Y at position409 of SEQ ID NO: 2 remains unchanged or is substituted with A, C, D, G,N, S, T or V; and iii. an amino acid P at position 410 of SEQ ID NO: 2remains unchanged or is substituted with A, C, G, I, L, M, N, S, T or V.7. The RNA polymerase variant according to claim 4, wherein the RNApolymerase variant is derived from Thermococcus gorgonarius DNApolymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2;and wherein: i. an amino acid L at position 408 of SEQ ID NO: 2 issubstituted with A, C, D, F, G, H, K, M, N, Q, S, W, or Y; ii. an aminoacid Y at position 409 of SEQ ID NO: 2 remains unchanged or issubstituted with A, C, D, G, N, S, T or V; iii. an amino acid P atposition 410 of SEQ ID NO: 2 remains unchanged or is substituted with A,C, G, I, L, M, N, S, T or V; and iv. an amino acid A at position 485 ofSEQ ID NO: 2 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q,R, T, V, W or Y.
 8. The RNA polymerase variant according to claim 4,wherein the RNA polymerase variant is derived from Thermococcuskodakarensis DNA polymerase (Kod1) having a wild-type amino acidsequence of SEQ ID NO: 3; and wherein: i. an amino acid L at position408 of SEQ ID NO: 3 is substituted with A, C, D, F, G, H, K, M, N, Q, S,W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 3 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; and iii. anamino acid P at position 410 of SEQ ID NO: 3 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.
 9. The RNA polymerasevariant according to claim 4, wherein the RNA polymerase variant isderived from Thermococcus kodakarensis DNA polymerase (Kod1) having awild-type amino acid sequence of SEQ ID NO: 3; and wherein: i. an aminoacid L at position 408 of SEQ ID NO: 3 is substituted with A, C, D, F,G, H, K, M, N, Q, S, W, or Y; ii. an amino acid Y at position 409 of SEQID NO: 3 remains unchanged or is substituted with A, C, D, G, N, S, T orV; iii. an amino acid P at position 410 of SEQ ID NO: 3 remainsunchanged or is substituted with A, C, G, I, L, M, N, S, T or V; and iv.an amino acid A at position 485 of SEQ ID NO: 3 is substituted with C,D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y.
 10. The RNApolymerase variant according to claim 4, wherein the RNA polymerasevariant is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase(9°N) having a wild-type amino acid sequence of SEQ ID NO: 4; andwherein: i. an amino acid L at position 408 of SEQ ID NO: 4 issubstituted with A, C, D, F, G, H, K, M, N, Q, S, W, or Y; ii. an aminoacid Y at position 409 of SEQ ID NO: 4 remains unchanged or issubstituted with A, C, D, G, N, S, T or V; and iii. an amino acid P atposition 410 of SEQ ID NO: 4 remains unchanged or is A, C, G, I, L, M,N, S, T or V.
 11. The RNA polymerase variant according to claim 4,wherein the RNA polymerase variant is derived from Thermococcus sp.(strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acidsequence of SEQ ID NO: 4; and wherein: i. an amino acid L at position408 of SEQ ID NO: 4 is substituted with A, C, D, F, G, H, K, M, N, Q, S,W, or Y; ii. an amino acid Y at position 409 of SEQ ID NO: 4 remainsunchanged or is r substituted with A, C, D, G, N, S, T or V; iii. anamino acid P at position 410 of SEQ ID NO: 4 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and iv. an amino acid Aat position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, I,K, L, M, N, P, Q, R, T, V, W or Y.
 12. The RNA polymerase variantaccording to claim 4, wherein the RNA polymerase variant is derived fromPyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acidsequence of SEQ ID NO: 5; and wherein: i. an amino acid L at position409 of SEQ ID NO: 5 is substituted with A, C, D, F, G, H, K, M, N, Q, S,W, or Y; ii. an amino acid Y at position 410 of SEQ ID NO: 5 remainsunchanged or is substituted with A, C, D, G, N, S, T or V; and iii. anamino acid P at position 411 of SEQ ID NO: 5 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V.
 13. The RNA polymerasevariant according to claim 4, wherein the RNA polymerase variant isderived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-typeamino acid sequence of SEQ ID NO: 5; and wherein: i. an amino acid L atposition 409 of SEQ ID NO: 5 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; ii. an amino acid Y at position 410 of SEQ ID NO: 5remains unchanged or is substituted with A, C, D, G, N, S, T or V; iii.an amino acid P at position 411 of SEQ ID NO: 5 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and iv. an amino acid Aat position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, I,K, L, M, N, P, Q, R, T, V, W or Y.
 14. The RNA polymerase variantaccording to claim 4, wherein the RNA polymerase variant is derived fromThermococcus litoralis DNA polymerase (Vent) having a wild-type aminoacid sequence of SEQ ID NO: 6; and wherein: i. an amino acid L atposition 411 of SEQ ID NO: 6 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; ii. an amino acid Y at position 412 of SEQ ID NO: 6remains unchanged or is substituted with A, C, D, G, N, S, T or V; andiii. an amino acid P at position 413 of SEQ ID NO: 6 remains unchangedor is substituted with A, C, G, I, L, M, N, S, T or V.
 15. The RNApolymerase variant according to claim 4, wherein the RNA polymerasevariant is derived from Thermococcus litoralis DNA polymerase (Vent)having a wild-type amino acid sequence of SEQ ID NO: 6; and wherein: i.an amino acid L at position 411 of SEQ ID NO: 6 is substituted with A,C, D, F, G, H, K, M, N, Q, S, W, or Y; ii. an amino acid Y at position412 of SEQ ID NO: 6 remains unchanged or is substituted with A, C, D, G,N, S, T or V; iii. an amino acid P at position 413 of SEQ ID NO: 6remains unchanged or is substituted with A, C, G, I, L, M, N, S, T or V;and iv. an amino acid A at position 488 of SEQ ID NO: 6 is substitutedwith C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y.
 16. The RNApolymerase variant according to claim 4, wherein the RNA polymerasevariant is derived from Methanosarcina acetivorans DNA polymerase (Mac)having a wild-type amino acid sequence of SEQ ID NO: 7; and wherein: i.an amino acid L at position 485 of SEQ ID NO: 7 is substituted with A,C, D, F, G, H, K, M, N, Q, S, W, or Y; ii. an amino acid Y at position486 of SEQ ID NO: 7 remains unchanged or is substituted with A, C, D, G,N, S, T or V; and iii. an amino acid P at position 487 of SEQ ID NO: 7remains unchanged or is substituted with A, C, G, I, L, M, N, S, T or V.17. The RNA polymerase variant according to claim 4, wherein the RNApolymerase variant is derived from Methanosarcina acetivorans DNApolymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7;and wherein: i. an amino acid L at position 485 of SEQ ID NO: 7 issubstituted with A, C, D, F, G, H, K, M, N, Q, S, W, or Y; ii. an aminoacid Y at position 486 of SEQ ID NO: 7 remains unchanged or issubstituted with A, C, D, G, N, S, T or V; iii. an amino acid P atposition 487 of SEQ ID NO: 7 remains unchanged or is substituted with A,C, G, I, L, M, N, S, T or V; and iv. an amino acid A at position 565 ofSEQ ID NO: 7 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q,R, T, V, W or Y.
 18. The RNA polymerase variant according to claim 4,wherein the RNA polymerase variant is derived from Pyrobaculumislandicum DNA polymerase (Pis) having a wild-type amino acid sequenceof SEQ ID NO: 8; and wherein: i. an amino acid M at position 426 of SEQID NO: 8 is substituted with A, C, D, F, G, H, K, N, Q, S, W, or Y; ii.an amino acid Y at position 427 of SEQ ID NO: 8 remains unchanged or issubstituted with A, C, D, G, N, S, T or V; and iii. an amino acid P atposition 428 of SEQ ID NO: 8 remains unchanged or is substituted with A,C, G, I, L, M, N, S, T or V.
 19. The RNA polymerase variant according toclaim 4, wherein the RNA polymerase variant is derived from Pyrobaculumislandicum DNA polymerase (Pis) having a wild-type amino acid sequenceof SEQ ID NO: 8; and wherein: i. an amino acid M at position 426 of SEQID NO: 8 is substituted with A, C, D, F, G, H, K, N, Q, S, W, or Y; ii.an amino acid Y at position 427 of SEQ ID NO: 8 remains unchanged or issubstituted with A, C, D, G, N, S, T or V; iii. an amino acid P atposition 428 of SEQ ID NO: 8 remains unchanged or is substituted with A,C, G, I, L, M, N, S, T or V; and iv. an amino acid A at position 508 ofSEQ ID NO: 8 is substituted with C, D, E, F, G, H, I, K, L, M, N, P, Q,R, T, V, W or Y.
 20. The RNA polymerase variant according to claim 4,wherein the RNA polymerase variant is derived from Sulfolobussolfataricus DNA polymerase (Sso) having a wild-type amino acid sequenceof SEQ ID NO: 9; and wherein: i. an amino acid L at position 518 of SEQID NO: 9 is substituted with A, C, D, F, G, H, K, M, N, Q, S, W, or Y;ii. an amino acid Y at position 519 of SEQ ID NO: 9 remains unchanged oris substituted with A, C, D, G, N, S, T or V; and iii. an amino acid Pat position 520 of SEQ ID NO: 9 remains unchanged or is substituted withA, C, G, I, L, M, N, S, T or V.
 21. The RNA polymerase variant accordingto claim 4, wherein the RNA polymerase variant is derived fromSulfolobus solfataricus DNA polymerase (Sso) having a wild-type aminoacid sequence of SEQ ID NO: 9; and wherein: i. an amino acid L atposition 518 of SEQ ID NO: 9 is substituted with A, C, D, F, G, H, K, M,N, Q, S, W, or Y; ii. an amino acid Y at position 519 of SEQ ID NO: 9remains unchanged or is substituted with A, C, D, G, N, S, T or V; iii.an amino acid P at position 520 of SEQ ID NO: 9 remains unchanged or issubstituted with A, C, G, I, L, M, N, S, T or V; and iv. an amino acid Aat position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, I,K, L, M, N, P, Q, R, T, V, W or Y.
 22. The RNA polymerase variant ofclaim 1, wherein the RNA polymerase variant exhibits an activity ofsynthesizing nucleic acids in a template-independent manner by adding atleast one nucleotide selected from the group of naturally occurringnucleotide, nucleotide analogue, or a mixture thereof, to an extendibleinitiator.
 23. The RNA polymerase variant of claim 22, wherein theextendible initiator comprises a single-stranded oligonucleotideinitiator, a blunt-ended double-stranded oligonucleotide initiator, or amixture thereof.
 24. The RNA polymerase variant of claim 22, wherein theextendible initiator is a free form nucleic acid to be reacted in aliquid phase.
 25. The RNA polymerase variant of claim 22, wherein theextendible initiator is immobilized on a solid support, wherein thesolid support comprises a particle, bead, slide, array surface,membrane, flow cell, well, microwell, nano-well, chamber, microfluidicchamber, channel, or microfluidic channel.
 26. The RNA polymerasevariant of claim 22, wherein the at least one nucleotide is linked witha detectable label.
 27. The RNA polymerase variant of claim 22, whereinthe at least one nucleotide comprises a ribose.
 28. The RNA polymerasevariant of claim 22, wherein the RNA polymerase variant exhibits theactivity at reaction temperatures ranging from 10° C. to 100° C.
 29. Akit for performing de novo enzymatic nucleic acid synthesis, comprisingan RNA polymerase variant derived from a wild-type B-family DNApolymerase having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, and 17, wherein the RNA polymerase variant exhibits activity ofsynthesizing nucleic acids in a template-independent manner by adding atleast one nucleotide selected from the group of naturally occurringnucleotide, nucleotide analogue, or a mixture thereof, to an extendibleinitiator, thereby synthesizing a desired nucleic acid sequence.
 30. Amethod for template-independent synthesis of an RNA oligonucleotide,comprising: (d) providing an initiator oligonucleotide, (e) providing anRNA polymerase variant; (f) combining the initiator oligonucleotide, theRNA polymerase variant and one or more nucleotides under conditionssufficient for the addition of at least one nucleotide to the 3′ end ofthe initiator oligonucleotide; wherein the RNA polymerase variantcomprising: a motif A, and a motif B corresponding respectively topositions 706 to 730, and 843 to 855 of a consensus sequence (SEQ IDNO:1); and at least one amino acid substitution at a position in themotif A, the motif B, or the combination thereof; wherein the RNApolymerase variant has a reduced or deficient 3′ to 5′ exonucleaseactivity.